Novel genes associated with the maintenance of differentiation of smooth muscle cells

ABSTRACT

A cDNA fragment participating in the maintenance of smooth muscle differentiation was isolated using a culture system of chicken gizzard smooth muscle cells, the differential display method and the subtracted hybridization method. Using the obtained cDNA sequence as a query, cDNA sequences of Helix Research Institute (Japanese Patent Application No. 2000-118776) were retrieved, and thus, a novel gene “C-NT2RP3001495” was obtained. The protein encoded by this gene has two WW domains that participate in protein interactions in the N-terminal domain. Evidence suggests that this protein binds to other proteins, and thus regulates the intracellular signal transduction, gene expression, and so on, thereby participating in the maintenance of the differentiation of smooth muscle cells. This protein and compounds regulating the expression thereof are markedly useful in developing drugs for various diseases associated with abnormality in the maintenance of smooth muscle cell differentiation.

[0001] This application is a continuation-in-part of PCT/JP00/05059,filed Jul. 28, 2000, which claims priority to U.S. ProvisionalApplication No. 60/159,590, filed Oct. 18, 1999, and No. 60/183,322,filed Feb. 17, 2000; and Japanese Patent Application Nos. 11-248036,filed Jul. 29, 1999; 2000-118776, filed Jan. 11, 2001; and 2000-183767,filed May 2, 2000.

TECHNICAL FIELD

[0002] The present invention relates to novel human proteins associatedwith the maintenance of differentiation of smooth muscle cells.

BACKGROUND

[0003] The smooth muscle cell is a major muscle cell in tissues such asblood vessel, trachea, digestive tract, urinary bladder, and uterus, andits important function is the regulation of contraction and relaxation.Recently, a relationship has been revealed between phenotypic modulationof the smooth muscle cells, wherein the cell looses the contractionability and thereby acquires proliferation ability, and the pathologicalstate. The proliferation of vascular smooth muscle cell is closelyassociated with the manifestation of the pathological state such asrestenosis occurring after percutaneous transluminal coronaryangioplasty (PTCA). It has been clarified that vascular tunica mediasmooth muscle cells acquire motility through phenotypic modulation to adedifferentiated type in the early stage of the onset ofarteriosclerosis, and thus, migrate to the vascular endothelium, whichis the major cause of hypertrophy of vascular endothelium. However,there are still many obscure points about the phenotypic modulation ofsmooth muscle cells, for example, associated genes, molecular mechanismthereof, and so on. A better understanding of how the smooth musclecells maintain the differentiated phenotype and how their phenotypes areconverted to the dedifferentiated type is needed to develop therapeuticmethods for morbid states caused by smooth muscle cell proliferation.The mechanism of the phenotypic modulation may be elucidated byanalyzing the genes associated with the maintenance of differentiatedtype of smooth muscle cells, such genes being applicable as therapeuticagents and diagnostic agents for diseases caused by the aberrantproliferation of smooth muscle cells; ischemic heart diseases such asarteriosclerosis, myocardial infarction, aortic aneurysm, and cerebralapoplexy; cerebral vascular disorders; and vascular dementia. Inaddition, glomerulonephritis, pulmonary fibrosis, cerebralarteriosclerosis, and hepatitis, which correspond to a state of aberrantproliferation of mesangial cells, alveolar epithelial cells, pericytes,and Ito cells, respectively—cells that have extremely similarcharacteristics to the smooth muscle cells—are presumed to be diseasescaused by cellular transformation occurring through a mechanism similarto that of smooth muscle cells; and thus, the genes therein may also beapplicable as therapeutic agent and diagnostic agent for these chronicdiseases.

SUMMARY

[0004] The object of the present invention is to provide novel proteinsassociated in the maintenance of differentiation of smooth muscle cells,genes encoding them, and production and use of the proteins and genes.

[0005] To accomplish the objects described above, the present inventorsvigorously carried out the following research. The present inventorsfirst constructed a cDNA library by subtracting cDNAs ofdedifferentiated smooth muscle cells derived from chicken gizzard fromcDNAs of differentiated smooth muscle cells derived from chickengizzard, in order to elucidate the mechanism for the maintenance ofdifferentiation of smooth muscle cells. The nucleotide sequence of theobtained cDNA fragment was determined, and thus a sequence (SEQ ID NO:3)named “12F08” was obtained. It has been revealed that “12F08” exhibits ahomology of 82% to the clone Hs#S1388556 belonging to a human Unigenecluster Hs.128045.

[0006] In the next step, the present inventors obtained the“Hs128045_(—)12F08con” sequence by preparing a contig via assemblingsequences belonging to the human Unigene cluster Hs.128045. The pfammotif database was then searched for the “Hs128045_(—)12F08con” byutilizing estwisedb in the database search program Wise2 designed byEwan Birney at Sanger Center. The results showed that each of 12F08 andHs128045_(—)12F08con contained two WW domains that are importantfunctional domains for protein-protein interaction.

[0007] Furthermore, the present inventors searched cDNA sequences ofHelix Research Institute (helix clones; Japanese Patent Application No.Hei 11-248036; Japanese Patent Application No. 2000-118776) forhomologues using the above-mentioned sequence, “Hs128045_(—)12F08con”,obtained from Unigene Cluster as a query. These helix clones are highlyexpected to have the full length sequence, which are obtained by thecombined use of: [1] preparation of cDNA library, which comprises cDNAhaving a full-length sequence at a high rate, by the oligo-cappingmethod; and [2] evaluation system for the cDNA to determine whether itcontains the full-length sequence based on the 5′ end sequence (theselection is achieved based on the evaluation using ATGpr aftereliminating non-full length clones as compared with an EST). The resultsof homology search showed that the query clone was identical to thehelix clone “C-T2RP3001495”. In addition, it was also revealed that thequery clone is identical to the gene for Hs.519 Human oxidoreductase(HHCMA56) of Unigene. However, the sequence of HHCMA56 contains readingmistakes of nucleotides, and thus it has been deposited as a geneencoding a protein consisting of 371 amino acids which is entirelydifferent from the protein of “C-NT2RP3001495”. Thus, it can be statedthat “C-NT2RP3001495” is a novel protein found for the first time by thepresent inventors. The “C-NT2RP3001495” is a protein consisting of 414amino acids, which has two WW domain sequences.

[0008] The present inventors then analyzed the expression level of gene“12F08” in a variety of tissues by real-time PCR. The results showedthat the gene was expressed at high levels in the differentiated smoothmuscle and gizzard, suggesting that “12F08” encodes a protein associatedwith the maintenance of differentiation of smooth muscle cells. Thus,human “C-NT2RP3001495” is presumed to be a protein associated with themaintenance of differentiation of smooth muscle cells.

[0009] The human “C-NT2RP3001495” is expected to be useful as apharmaceutical for diseases caused by the aberrant proliferation ofsmooth muscle cells; ischemic heart diseases such as arteriosclerosis,myocardial infarction, aortic aneurysm, cerebral apoplexy; cerebralvascular disorders; and vascular dementia; as well as forglomerulonephritis, pulmonary fibrosis, cerebral arteriosclerosis, andhepatitis, which correspond to the states of aberrant proliferation ofmesangial cells, alveolar epithelial cells, pericytes, and Ito cells,respectively—cells which have extremely similar characteristics to thesmooth muscle cells.

[0010] As described above, the present inventors found novel proteinsassociated with the maintenance of differentiation of smooth musclecells, and thereby accomplished the present invention.

[0011] Specifically, the present invention relates to novel proteinswhich participate in the maintenance of differentiation of smooth musclecells, genes encoding the proteins, and production and uses of theproteins and genes. More specifically, the present invention providesthe following:

[0012] [1] a DNA of any one of the following (a) to (d):

[0013] (a) a DNA encoding a protein consisting of the amino acidsequence of SEQ ID NO:2,

[0014] (b) a DNA comprising the coding region of the nucleotide sequenceof SEQ ID NO:1,

[0015] (c) a DNA encoding a protein which (i) comprises the amino acidsequence of SEQ ID NO:2 in which one or more amino acids aresubstituted, deleted, inserted and/or added, and (ii) is functionallyequivalent to the protein consisting of the amino acid sequence of SEQID NO:2, and

[0016] (d) a DNA hybridizing under a stringent condition to a DNAconsisting of the nucleotide sequence of SEQ ID NO:1, which encodes aprotein functionally equivalent to the protein consisting of the aminoacid sequence of SEQ ID NO:2;

[0017] [2] a DNA encoding a partial peptide of a protein consisting ofthe amino acid sequence of SEQ ID NO:2;

[0018] [3] a protein or peptide encoded by the DNA of [1] or [2];

[0019] [4] a vector into which the DNA of [1] or [2] has been inserted;

[0020] [5] a host cell containing the DNA of [1] or [2], or the vectorof [4];

[0021] [6] a method for producing the protein or peptide of [3], whichcomprises the steps of culturing the host cell of [5], and recoveringthe expressed protein from the host cell or the culture supernatant;

[0022] [7] an antibody binding to the protein of [3];

[0023] [8] a polynucleotide containing at least 15 nucleotidescomplementary to a DNA consisting of the nucleotide sequence of SEQ IDNO:1 or the complementary strand thereof; and

[0024] [9] a method of screening for a compound that binds to theprotein of [3], which comprises the steps of:

[0025] (a) contacting a test sample containing at least one compoundwith the protein or a partial peptide thereof,

[0026] (b) detecting the binding activity of the compound with theprotein or a partial peptide thereof, and

[0027] (c) selecting the compound that has the activity of binding tothe protein or a partial peptide thereof.

[0028] The present invention provides a human-derived gene“C-NT2RP3001495” that encodes a novel protein which participates in themaintenance of differentiation of smooth muscle cells. The nucleotidesequence of human-derived cDNA “C-NT2RP3001495” is shown in SEQ ID NO:1,and the amino acid sequence encoded by the cDNA is shown in SEQ ID NO:2.As seen in SEQ ID NO:1, the human cDNA “C-NT2RP3001495” has an ORFencoding a protein consisting of 414 amino acids.

[0029] The inventive human “C-NT2RP3001495” gene has been selected as ahelix clone (Japanese Patent Application No. Hei 11-248036; JapanesePatent Application No. 2000-118776) exhibiting homology to the cDNAfragment “12F08”, isolated as a gene fragment associated with themaintenance of smooth muscle differentiation by using culture system ofchicken gizzard smooth muscle cells. High expression of theabove-mentioned “12F08” was observed in differentiated smooth muscle andgizzard. Further, the results of a motif search showed that the proteincontained two WW domains in the N-terminal region that participate inprotein-protein interaction. This suggests that the protein binds toother proteins and regulates intracellular signal transduction, geneexpression or the like, and thereby participates in the maintenance ofdifferentiation of smooth muscle cells. Further, the human“C-NT2RP3001495” protein of the present invention has an Adh short motifthat is found in oxidoreductases and dehydrases, and therefore, it ispotentially an oxidoreductase or dehydrase itself.

[0030] The expression pattern and structural properties of human“C-NT2RP3001495” suggest that it serves an important function in livingbody, and thus, it can be a useful target for drug development. Inaddition, compounds binding to human “C-NT2RP3001495” and compoundsregulating human “C-NT2RP3001495” gene expression are expected to beapplicable for the development of prophylactic or therapeutic agents fora variety of diseases caused by abnormalities in the maintenance ofdifferentiation of smooth muscle cells.

[0031] Further the present invention includes proteins functionallyequivalent to the human “C-NT2RP3001495” protein (SEQ ID NO:2). Suchproteins include, for example, mutants, homologues, and variants of thehuman “C-NT2RP3001495” protein. The term “functionally equivalent”herein means that the protein of interest has a function associated withthe maintenance of differentiation of smooth muscle cells like that ofthe “C-NT2RP3001495” protein. Specifically, the smooth muscle cells canbe divided into two types, namely differentiated and dedifferentiatedtypes thereof, by utilizing the expression of genes, such as theexpression of calponin gene or h-caldesmon gene, which arecharacteristic of regulations at the transcription level and at the mRNAsplicing level that are known as characteristics of the differentiatedstate (Cell Technology, 16(10):1496 (1997)). The levels of geneexpression are compared between the differentiated cells anddedifferentiated cells prepared from the smooth muscle cells in whichgenes characteristic of the differentiated type are expressed. When theexpression level of a gene is significantly higher or alternativelylower in the differentiated smooth muscle cells than in dedifferentiatedcells, then it can be determined that it is highly probable that thegene has a function associated with the maintenance of differentiation.For example, chicken gizzard smooth muscle cells are included in smoothmuscle cells in which genes characteristic of the differentiated typeare expressed.

[0032] One method for preparing functionally equivalent proteins wellknown to those skilled in the art involves the introduction of mutationsinto the proteins. For example, one skilled in the art can prepareproteins functionally equivalent to the human “C-NT2RP3001495” protein(SEQ ID NO:2) by introducing appropriate mutations into the amino acidsequence of the protein using the site-directed mutagenesis method(Hashimoto-Gotoh, et al., Gene 152:271-275, 1995; Zoller et al., MethodsEnzymol. 100:468-500, 1983; Kramer, et al., Nucleic Acids Res.12:9441-9456, 1984; Kramer et al., Methods. Enzymol. 154:350-367, 1987;Kunkel, Proc. Natl. Acad. Sci. USA. 82:488-492, 1985; Kunkel, MethodsEnzymol. 85:2763-2766, 1988) and such. Mutation of amino acids may occurin nature, too. The proteins of the present invention include proteinscomprising the amino acid sequence of human “C-NT2RP3001495” protein(SEQ ID NO:2) in which one or more amino acids are mutated, so long asthe resulting mutant protein is functionally equivalent to the protein.In such a mutant protein, the number of the amino acids to be mutated isusually 50 residues or less, preferably 30 residues or less, and morepreferably 10 residues or less (e.g., 5 residues or less).

[0033] The amino acid residue to be mutated is preferably mutated into adifferent amino acid that allows the properties of the amino acidside-chain to be conserved. Examples of properties of amino acid sidechains include: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and aminoacids comprising the following side chains: an aliphatic side-chain (G,A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); asulfur atom containing side-chain (C, M); a carboxylic acid and amidecontaining side-chain (D, N, E, Q); a base containing side-chain (R, K,H); and an aromatic containing side-chain (H, F, Y, W) (The parentheticletters indicate the one-letter codes of amino acids).

[0034] It is well known that a protein having deletion, addition, and/orsubstitution of one or more amino acid residues in the sequence of aprotein can retain the original biological activity (Mark et al., Proc.Natl. Acad. Sci. U.S.A. 81:5662-5666, 1984; Zoller et al., Nucleic AcidsRes. 10:6487-6500, 1982; Wang et al., Science 224:1431-1433;Dalbadie-McFarland et al. Proc. Natl. Acad. Sci. U.S.A. 79:6409-6413,1982).

[0035] A protein having the amino acid sequence of human“C-NT2RP3001495” protein to which one or more amino acid residues havebeen added, is exemplified by a fusion protein containing the human“C-NT2RP3001495” protein. Fusion proteins, in which the human“C-NT2RP3001495” protein is fused to other peptides or proteins, areincluded in the present invention. Fusion proteins can be made usingtechniques well known to those skilled in the art, for example, bylinking the DNA encoding the human “C-NT2RP3001495” protein (SEQ IDNO:2) in frame with the DNA encoding other peptides or proteins,followed by inserting the DNA into an expression vector and expressingit in a host. There is no restriction as to the peptides or proteins tobe fused to the protein of the present invention.

[0036] For instance, known peptides which may be used for the fusioninclude the FLAG peptide (Hopp et al., BioTechnology 6:1204-1210, 1988),6×His that is made up of six histidine residues, 10×His, influenzahemagglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIVfragment, T7-tag, HSV-tag, E-tag, SV40 T antigen fragment, lck tag,α-tubulin fragment, B-tag, and Protein C fragment. Also,glutathione-S-transferase (GST), influenza hemagglutinin (HA), theconstant region of immunoglobulin, β-galactosidase, maltose bindingprotein (MBP), and the like may be used as a protein to be fused withthe protein of this invention. Fusion proteins can be prepared by fusingthe DNA encoding these peptides or proteins, which are commerciallyavailable, with the DNA encoding the protein of the invention, andexpressing the fused DNA.

[0037] An alternative method for preparing functionally equivalentproteins known to those skilled in the art utilizes, for example, thehybridization technique (Sambrook et al., Molecular Cloning 2nd ed.9.47-9.58, Cold Spring Harbor Lab. Press, 1989). Generally, one skilledin the art can isolate DNAs highly homologous to the whole or part ofthe DNA sequence encoding the human “C-NT2RP3001495” protein (SEQ IDNO:1), and then isolate proteins functionally equivalent to the human“C-NT2RP3001495” protein based on those DNAs isolated. The presentinvention includes proteins that are (i) encoded by a DNA hybridizing toa DNA encoding the protein of human “C-NT2RP3001495” and (ii)functionally equivalent to the human protein of “C-NT2RP3001495”. Suchproteins include, for example, homologues derived from human and otheranimals (for example, protein encoded by a DNA from mouse, rat, rabbit,cattle, chicken, etc.). The protein (SEQ ID NO:4) encoded by “12F08” maybe exemplified as the homologue from chicken.

[0038] Those skilled in the art can properly select hybridizationconditions to be used for the isolation of DNAs encoding proteinsfunctionally equivalent to the human protein of “C-NT2RP3001495”.Hybridization conditions include low stringent conditions. Low stringentconditions may be, for example, 42° C. in 2×SSC and 0.1% SDS, preferably50° C. in 2×SSC and 0.1% SDS for washing after hybridization. Morepreferably, high stringent conditions such as 65° C. in 0.1×SSC and 0.1%SDS may be chosen. DNA with higher homology may be efficiently obtainedat higher temperature under these conditions. However, several factorsare thought to influence the stringency of hybridization, such astemperatures and salt concentrations, and one skilled in the art cansuitably select these factors to accomplish a similar stringency. Moreguidelines for the hybridization condition are available in the art, forexample, in a reference by Sambrook et al. (1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, N.Y.) and in unit 2.10 ofthe reference by Ausubel et al. (1995, Current Protocols in MolecularBiology, John Wiley & Sons, N.Y.).

[0039] Also, in lieu of hybridization, it is also possible to isolatefunctionally equivalent proteins by a gene amplification method, such asPCR, by synthesizing sequences based on the sequence information of theDNA encoding the human “C-NT2RP3001495” protein (SEQ ID NO:1) and usingthem as primers.

[0040] The proteins functionally equivalent to the human“C-NT2RP3001495” proteins encoded by the DNA isolated by thehybridization or gene amplification techniques, usually are highlyhomologous to the human “C-NT2RP3001495” proteins (SEQ ID NO:2) at theamino acid sequence level. The proteins of the invention includeproteins functionally equivalent to the human “C-NT2RP3001495” proteinand are highly homologous to the amino acid sequence of SEQ ID NO:2.“Highly homologous” means typically 65% or higher, preferably 75% orhigher, more preferably 85% or higher, and even more preferably 95% orhigher identity at the amino acid level. Homology between proteins canbe determined according to the algorithm described in the literature(Wilbur et al., Proc. Natl. Acad. Sci. USA 80:726-730, 1983).

[0041] The proteins of the present invention may have variations in theamino acid sequence, molecular weight, isoelectric point, presence orabsence of sugar chains, or form, depending on the cell or host used toproduce them or the purification method utilized as described below.Nevertheless, so long as the protein obtained has a function equivalentto the human “C-NT2RP3001495” protein, it is within the scope of thepresent invention. For example, when the inventive protein is expressedin prokaryotic cells, e.g., E. coli, a methionine residue is added atthe N-terminus of the original protein. The present invention alsoincludes such proteins.

[0042] The proteins of the present invention can be prepared asrecombinant proteins or as naturally occurring proteins, using methodscommonly known in the art. The recombinant protein can be, for example,prepared as follows. The DNA encoding the protein of this invention(e.g., DNA having the nucleotide sequence of SEQ ID NO:1) is insertedinto an appropriate expression vector, and introduced into suitable hostcells. Subsequently, the resulting transformants, the host cell insertedwith the expression vector, are recovered, extracted and then purifiedby chromatography utilizing ion exchange, reverse phase, or gelfiltration, or by affinity chromatography with a column in which theantibodies against the protein of the present invention are fixed, or bya combination of these columns.

[0043] Alternatively, the protein of the invention can be prepared byexpressing the protein in host cells (e.g., animal cells or E. coli) asa fusion protein with glutathione S transferase protein, or as arecombinant protein with multiple histidine residues. The expressedprotein can be purified using a glutathione column or nickel column.Subsequently, if necessary, regions of the fusion protein (apart fromthe desired protein) can be digested and removed with thrombin, factorXa, etc.

[0044] The natural protein corresponding to the protein of the inventioncan be isolated by methods well known in the art, for example, bypurifying tissue or cell extracts containing a protein of the inventionwith an affinity column to which the antibody that binds to the proteinof the present invention described below is bound. The antibody may be apolyclonal antibody or monoclonal antibody.

[0045] The term “substantially pure” as used herein in reference to agiven polypeptide means that the polypeptide is substantially free fromother biological macromolecules. For example, the substantially purepolypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight.Purity can be measured by any appropriate standard method known in theart, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

[0046] Accordingly, the invention includes a polypeptide having asequence shown as SEQ ID NO:2. The invention also includes apolypeptide, or fragment thereof, that differs from the correspondingsequence shown as SEQ ID NO:2. The differences are, preferably,differences or changes at a non-essential residue or a conservativesubstitution. In one embodiment, the polypeptide includes an amino acidsequence at least about 60% identical to a sequence shown as SEQ IDNO:2, or a fragment thereof. Preferably, the polypeptide is at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ IDNO:2 and has at least one cell differentiation-related function oractivity described herein, e.g., the polypeptide is involved in themaintenance of differentiation of smooth muscle cells. Preferredpolypeptide fragments of the invention are at least 10%, preferably atleast 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of thesequence shown as SEQ ID NO:2 and have at least one celldifferentiation-related function or activity described herein.Alternatively, the fragment can be merely an immunogenic fragment.

[0047] The present invention also includes partial peptides of theproteins of the present invention. The partial peptides of the presentinvention comprise at least 7 or more amino acids, preferably 8 or moreamino acids, more preferably 9 or more amino acids. The partial peptidescan be used, for example, for generating antibodies against the proteinof the present invention, screening of compounds binding to the proteinof the present invention, or screening of promoters or inhibitors forthe protein of the present invention. The partial peptides can be usedas antagonists or competitive inhibitors for the protein of thisinvention. The partial peptides of the invention can be produced bygenetic engineering, known methods of peptide synthesis, or by digestingthe protein of the invention with an appropriate peptidase. For peptidesynthesis, for example, solid phase synthesis or liquid phase synthesismay be used.

[0048] DNA encoding an inventive protein can be used for the productionof the inventive protein in vivo and in vitro as described above; it isalso applicable to, for example, gene therapy for diseases caused by theabnormality in the gene encoding the inventive protein and for diseasesthat can be treated by the inventive protein. Any type of DNA, such ascDNA synthesized from mRNA, genomic DNA or synthetic DNA, can be used solong as the DNA encodes a protein of the present invention. Also so longas they can encode a protein of the present invention, DNAs comprisingarbitrary sequences based on the degeneracy of the genetic code are alsoincluded.

[0049] The DNA of the present invention can be prepared using methodsknown in the art. For example, a cDNA library can be constructed fromthe cells expressing the protein of the present invention, andhybridization can be conducted using a part of the DNA sequence of thepresent invention (for example, SEQ ID NO:1) as a probe. cDNA librariesmay be prepared by, for example, the method described in the literature(Sambrook et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress, 1989), and also, commercially available ones can be used.Alternatively, the DNA of the present invention can be obtained bypreparing the RNA from the cells expressing the protein of the presentinvention, synthesizing cDNA by reverse transcriptase, synthesizing theoligo-DNAs based on the DNA sequence of the present invention (forexample, SEQ ID NO:1), and amplifying the cDNA encoding the protein ofthe present invention by PCR using the oligonucleotides as primers.

[0050] As used herein, an “isolated nucleic acid” is a nucleic acid, thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

[0051] Accordingly, in one aspect, the invention provides an isolated orpurified nucleic acid molecule that encodes a polypeptide describedherein or a fragment thereof Preferably, the isolated nucleic acidmolecule includes a nucleotide sequence that is at least 60% identicalto the nucleotide sequence shown in SEQ ID NO:1. More preferably, theisolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to thenucleotide sequence shown in SEQ ID NO:1. In the case of an isolatednucleic acid molecule which is longer than or equivalent in length tothe reference sequence, e.g., SEQ ID NO: 1, the comparison is made withthe full length of the reference sequence. Where the isolated nucleicacid molecule is shorter that the reference sequence, e.g., shorter thanSEQ ID NO: 1, the comparison is made to a segment of the referencesequence of the same length (excluding any loop required by the homologycalculation).

[0052] As used herein, “% identity” of two amino acid sequences, or oftwo nucleic acid sequences, is determined using the algorithm of Karlinand Altschul (PNAS USA 87:2264-2268, 1990), modified as in Karlin andAltschul, PNAS USA 90:5873-5877, 1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performedwith the NBLAST program, score=100, wordlength=12. BLAST proteinsearches are performed with the XBLAST program, score=50, wordlength=3.To obtain gapped alignment for comparison purposes GappedBLAST isutilized as described in Altschul et al. (Nucleic Acids Res.25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used to obtain nucleotide sequences homologous to a nucleic acidmolecule of the invention.

[0053] The nucleotide sequence of the obtained cDNA is determined tofind an open reading frame, and thereby the amino acid sequence of theprotein of the invention can be obtained. The cDNA obtained may also beused as a probe for screening a genomic library to isolate a genomicDNA.

[0054] More specifically, mRNAs may first be prepared from a cell,tissue, or organ in which the protein of the invention is expressed(e.g. tissues such as liver and kidney). Known methods can be used toisolate mRNAs; for instance, total RNA can be prepared by guanidineultracentrifugation (Chirgwin et al., Biochemistry 18:5294-5299, 1979)or the AGPC method (Chomczynski et al., Anal. Biochem. 162:156-159,1987). mRNA may then be purified from total RNA using mRNA PurificationKit (Pharmacia) and such; alternatively, mRNA may be directly purifiedby QuickPrep mRNA Purification Kit (Pharmacia).

[0055] The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized by using a kit such as the AMVReverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).Alternatively, cDNA may be synthesized and amplified following the5′-RACE method (Frohman et al., Proc. Natl. Acad. Sci. U.S.A.85:8998-9002, 1988; Belyavsky et al., Nucleic Acids Res. 17:2919-2932,1989) which uses primers described herein, the 5′-Ampli FINDER RACE Kit(Clontech), and polymerase chain reaction (PCR).

[0056] A desired DNA fragment is prepared from the PCR products andligated with a vector DNA. The recombinant vectors are used to transformE. coli and such, and a desired recombinant vector is prepared from aselected colony. The nucleotide sequence of the desired DNA is verifiedby conventional methods, such as dideoxynucleotide chain termination.

[0057] A DNA of the invention may be designed to have a sequence that isexpressed more efficiently by taking into account the frequency of codonusage in the host to be used for expression (Grantham et al., NucleicAcids Res. 9:43-74, 1981). The DNA of the present invention may bealtered by a commercially available kit or a conventional method. Forinstance, the DNA may be altered by digestion with restriction enzymes,insertion of a synthetic oligonucleotide or an appropriate DNA fragment,addition of a linker, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA, or TAG).

[0058] The DNA of the present invention also include a DNA hybridizingto a DNA consisting of the nucleotide sequence of SEQ ID NO:1 andencoding a protein functionally equivalent to the above-mentionedprotein of the present invention. Those skilled in the art can properlyselect the appropriate hybridization conditions, and specifically theabove-mentioned conditions can be used. Under these conditions, thehigher the temperature, the higher the homology of the obtained DNA willbe. The above-mentioned hybridizing DNA is preferably a naturallyoccurring DNA, for example, cDNA or chromosomal DNA.

[0059] The present invention also provides a vector into which a DNA ofthe present invention is inserted. The vectors of the present inventionare useful for maintaining the DNA of the present invention within hostcells or expressing the protein of the invention.

[0060] When the E. coli is used as a host cell, there is no limitationother than that the vector should have an “ori” to amplify andmass-produce the vector in E. coli (e.g., JM109, DH5α, HB 101, orXL1Blue), and a marker gene for selecting the transformed E. coli (e.g.,a drug-resistance gene selected by a drug such as ampicillin,tetracycline, kanamycin, or chloramphenicol). For example, M13-seriesvectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, and suchcan be used. pGEM-T, pDIRECT, pT7, and so on can also be used forsubcloning and excision of the cDNA as well as the vectors describedabove. When a vector is used to produce a protein of the presentinvention, an expression vector is especially useful. The expressionvector, for example, to be expressed in E. coli should have the abovecharacteristics to be amplified in E. coli. When E. coli, such as JM109,DH5α, HB101, or XL1 Blue, is used as the host cell, the vector shouldhave a promoter such as lacZ promoter (Ward et al., Nature 341:544-546,1989; FASEB J. 6:2422-2427, 1992), araB promoter (Better et al., Science240:1041-1043, 1988), or T7 promoter that can efficiently promote theexpression of the desired gene in E. coli. Other examples of the vectorsare pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, and pET(for this vector, BL21, a strain expressing T7 RNA polymerase, ispreferably used as the host).

[0061] Further, the vector may contain a signal sequence for thesecretion of polypeptides. The pelB signal sequence (Lei et al., J.Bacteriol. 169:4379, 1987) can be used as a signal sequence forsecretion of proteins, when the proteins are intended to be produced inthe periplasm of E. coli. Introduction of the vector into a host cellcan be performed, for example, by the calcium chloride method orelectroporation.

[0062] In addition to the vectors for E. coli, for example, the vectorfor producing the proteins of this invention may be a mammal-derivedexpression vector (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic AcidsRes. 18(17):5322, 1990), pEF, and pCDM8), an insect cell-derivedexpression vector (e.g., “Bac-to-BAC baculovairus expression system”(GibcoBRL) and pBacPAK8), a plant-derived expression vector (e.g., pMH1and pMH2), an animal virus-derived expression vector (e.g., pHSV, pMV,and pAdexLcw), a retrovirus-derived expression vector (e.g., pZIPneo),an yeast-derived expression vector (e.g., “Pichia Expression Kit”(Invitrogen), pNV11, and SP-Q01), a Bacillus subtilis-derived expressionvector (e.g., pPL608 and pKTH50).

[0063] In order to express proteins in animal cells, such as CHO, COS,and NIH3T3 cells, the vector should have a promoter necessary forexpression in such cells, e.g., SV40 promoter (Mulligan et al., Nature277:108, 1979), MMLV-LTR promoter, EF1α promoter (Mizushima et al.,Nucleic Acids Res. 18:5322, 1990), CMV promoter, etc., and morepreferably it has a marker gene for selecting transformants (forexample, a drug resistance gene selected by a drug (e.g., neomycin,G418, etc.)). Examples of vectors with these characteristics includepMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, and so on.

[0064] The method using CHO cells deficient in nucleic acid syntheticpathways as the host, and incorporating a vector (such as PCHOI) with aDHFR gene that compensates for the deficiency and amplifying the vectorwith methotrexate (MTX) can be mentioned as an example method for stablyexpressing a gene and amplifying the copy number in cells. And as amethod for transient expression, a method transforming the COS cells,which have the gene for SV40 T antigen on the chromosome, with a vector(such as pcDNA3) having the SV40 replication origin can be mentioned.The origin used for replication may be those of polyomavirus,adenovirus, bovine papilloma virus (BPV), and the like. In addition, theexpression vector may include a selection marker gene for amplificationof the gene copies in host cells. Examples of such markers include, butare not limited to, the aminoglycoside transferase (APH) gene, thethymidine kinase (TK) gene, the E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, and the dihydrofolate reductase (dhfr) gene.

[0065] The DNA of the present invention can be expressed in animals by,for example, inserting a DNA of the invention into an appropriate vectorand introducing the vector into a living body by the retrovirus method,liposome method, cationic liposome method, adenovirus method, and so on.Thus, gene therapy can be conducted for diseases caused by mutations inthe “C-NT2RP3001495” gene of this invention. The vectors used include,but are not limited to, adenoviral vectors (e.g., pAdexlcw) andretroviral vectors (e.g., pZIPneo). General techniques for genemanipulation, such as insertion of the DNA of the invention into avector, can be performed according to conventional methods (MolecularCloning, 5.61-5.63). The DNA of this invention can be administered tothe living body by an ex vivo method or in vivo method.

[0066] The present invention also provides a host cell into which thevector of the present invention has been introduced. The host cell intowhich the vector of the invention is introduced is not particularlylimited. E. coli and various animal cells can be used. The host cell ofthis invention can be used as, for example, a production system forproducing or expressing the protein of the invention. The productionsystem for producing a protein of the invention may be both in vitro orin vivo production system. For in vitro production, eukaryotic cells orprokaryotic cells can be used.

[0067] Useful eukaryotic host cells may be animal, plant, or fungicells. As animal cells, mammalian cells such as CHO (J. Exp. Med.108:945, 1995), COS, 3T3, myeloma, baby hamster kidney (BHK), HeLa, orVero cells, amphibian cells such as Xenopus oocytes (Valle et al. Nature291:340-358, 1981), or insect cells such as Sf9, Sf21, or Tn5 cells canbe used. CHO cells lacking DHFR gene (dhfr-CHO) (Proc. Natl. Acad. Sci.U.S.A. 77:4216-4220, 1980) or CHO K-1 (Proc. Natl. Acad. Sci. U.S.A.60:1275, 1968) may also be used. Among the animal cells, CHO cells areparticularly preferable for high-level expression. The vector can beintroduced into the host cell by, for example, the calcium phosphatemethod, the DEAE-dextran method, cationic liposome DOTAP (BoehringerMannheim) method, electroporation, lipofection, etc.

[0068] As plant cells, for example, plant cells originating fromNicotiana tabacum are known as protein production system and may be usedas callus cultures. As fungi cells, yeast cells such as Saccharomyces,including Saccharomyces cerevisiae, or filamentous fungi such asAspergillus, including Aspergillus niger, are known.

[0069] Useful prokaryotic cells include bacterial cells, such as E.coli, for example, JM109, DH5 α, and HB101, or Bacillus subtilis.

[0070] These cells are transformed by a desired DNA, and the resultingtransformants are cultured in vitro to obtain the protein. Transformantscan be cultured using known methods. Culture medium such as DMEM, MEM,RPMI1640, or IMDM may be used for animal cells. The culture medium canbe used with or without serum supplement such as fetal calf serum (FCS).The pH of the culture medium is preferably between about 6 and 8. Cellsare typically cultured at about 30 to 40° C. for about 15 to 200 hr, andthe culture medium may be replaced, aerated, or stirred if necessary.

[0071] Animal and plant hosts may be used for in vivo production. Forexample, a desired DNA can be introduced into an animal or plant host.Encoded proteins are produced in vivo, and then are recovered. Theseanimal and plant hosts are included in host cells of the presentinvention.

[0072] Animals to be used for the production system described aboveinclude mammals and insects. Mammals such as goat, porcine, sheep,mouse, and bovine may be used (Vicki Glaser, SPECTRUM BiotechnologyApplications (1993)). Alternatively, the mammals may be transgenicanimals.

[0073] For instance, a desired DNA may be prepared as a fusion gene,fused with a gene such as goat β casein gene which encodes a proteinspecifically produced into milk. DNA fragments comprising the fusiongene are injected into goat embryos, which are then transplanted back tofemale goats. Proteins of interest can be recovered from milk producedby the transgenic goats (i.e., those born from the goats that hadreceived the embryos) or from their offspring. To increase the amount ofmilk containing the proteins produced by transgenic goats, hormones maybe appropriately administered to them (Ebert et al.,Bio/Technology12:699-702, 1994).

[0074] Alternatively, insects, such as the silkworm, may be used.Baculoviruses into which the DNA encoding the protein of interest isinserted can be used to infect silkworms, and the desired protein can berecovered from their body fluid (Susumu et al., Nature 315:592-594,1985).

[0075] As plants, for example, tobacco can be used. In use of tobacco,DNA encoding the protein of interest may be inserted into a plantexpression vector, such as pMON530, which is introduced into bacteria,such as Agrobacterium tumefaciens. Then the bacteria is used to infecttobacco, such as Nicotiana tabacum, and a desired polypeptide can berecovered from their leaves (Julian et al., Eur. J. Immunol. 24:131-138,1994).

[0076] A protein of the present invention obtained as above may beisolated from inside or outside of the host cells (e.g., culture media),and purified as a substantially pure homogeneous protein. The method forprotein isolation and purification is not limited to any specificmethod; in fact, any standard method may be used. For instance, columnchromatography, filter, ultrafiltration, salt precipitation, solventprecipitation, solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric pointelectrophoresis, dialysis, recrystallization, and so on may beappropriately selected and combined to isolate and purify the protein.

[0077] For example, affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtration, reversephase chromatography, adsorption chromatography, and such may be usedfor chromatography (Daniel R. Marshak et al., Strategies for ProteinPurification and Characterization: A Laboratory Course Manual. Ed., ColdSpring Harbor Laboratory Press, 1996). These chromatographies may beperformed by liquid chromatography such as HPLC and FPLC. Thus, thepresent invention includes highly purified proteins, purified by theabove methods.

[0078] A protein of the present invention may be optionally modified orpartially deleted by treating it with an appropriate proteinmodification enzyme before or after purification. Useful proteinmodification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, glucosidase, and soon.

[0079] The present invention also provides antibodies that bind to theprotein of the invention. The antibody of the invention may take anyform, including monoclonal antibody, as well as polyclonal antibodies.Furthermore, antiserum obtained by immunizing an animal such as rabbitwith the protein of the invention, all classes of polyclonal andmonoclonal antibodies, human antibodies, and humanized antibodiesproduced by genetic recombination are included.

[0080] A protein of the invention used as the antigen to obtainantibodies may be derived from any animal species, but preferably it isderived from a mammal, such as a human, mouse, or rat, and morepreferably from human. A human-derived protein may be obtained from thenucleotide or amino acid sequences disclosed herein.

[0081] Herein, a protein used as an antigen may be a complete protein orpartial peptides thereof. A partial peptide may be, for example, anamino (N)-terminal or carboxy (C)-terminal fragment of the protein.Herein, an antibody is defined as an antibody that reacts with eitherthe full-length or a fragment of the protein.

[0082] A gene encoding a protein of the invention or its fragment may beinserted into a known expression vector, which is used to transform ahost cell as described herein. The desired protein or its fragment maybe recovered from the outside or inside of the host cell by any standardmethod, and may be used as an antigen. Alternatively, cells expressingthe protein or their lysates, or a chemically synthesized protein may beused as an antigen. Short peptides are preferably used as antigens byappropriately combining them with carrier proteins such as keyholelimpet hemocyanin, bovine serum albumin, and ovalbumin.

[0083] Any mammalian animal may be immunized with the antigen, butpreferably the compatibility with parental cells used for cell fusion istaken into account. In general, animals of Rodentia, Lagomorpha, orPrimates are used.

[0084] Animals of Rodentia include, for example, mouse, rat, andhamster. Animals of Lagomorpha include, for example, rabbit. Animals ofPrimates include, for example, a monkey of Catarrhini (old world monkey)such as crab-eating monkey, rhesus monkey, sacred baboon, or chimpanzee.

[0085] Methods for immunizing animals with antigens are known in theart. For instance, intraperitoneal injection or subcutaneous injectionof antigens is used as a standard method for immunization of mammals.More specifically, antigens may be diluted and suspended in anappropriate amount with phosphate buffered saline (PBS), physiologicalsaline, etc. If desired, the antigen suspension may be mixed with anappropriate amount of a standard adjuvant, such as Freund's completeadjuvant, made into emulsion, and then administered to mammals.Preferably, it is followed by several administrations of antigen mixedwith an appropriately amount of Freund's incomplete adjuvant every 4 to21 days. An appropriate carrier may also be used for immunization. Afterimmunization as above, serum is examined for increase of the amount ofdesired antibodies by a standard method.

[0086] Polyclonal antibodies against the proteins of the presentinvention may be prepared by collecting blood from the immunized mammalexamined for the increase of desired antibodies in the serum, and byseparating serum from the blood by any conventional method. Serumcontaining the polyclonal antibodies, or if necessary, a fractioncontaining the polyclonal antibodies may be isolated from the serum tobe used as the polyclonal antibodies of the present invention. Forexample, immunoglobulin G or M can be prepared by using an affinitycolumn coupled with the protein of the invention to obtain the fractionexclusively recognizing the protein of the invention, and then,purifying the fraction by using protein A or protein G column.

[0087] To prepare monoclonal antibodies, immune cells are collected fromthe mammal immunized with the antigen and checked for the increasedlevel of desired antibodies in the serum as described above, and aresubjected to cell fusion. The immune cells used for cell fusion arepreferably obtained from spleen. The other parent cell which is fusedwith the above immune cell is preferably a mammalian myeloma cell, andmore preferably a myeloma cell that has acquired a special feature thatcan be used for selection of fusion cells by drugs.

[0088] Cell fusion of the above immune cell and myeloma cell may beperformed by any standard method, such as those described in theliterature (Galfre et al., Methods Enzymol. 73:3-46, 1981).

[0089] Hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin, and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the time being sufficient to allow all the other cells,except desired hybridoma (non-fused cells), to die. Then, the standardlimiting dilution is performed to screen and clone a hybridoma cellproducing the desired antibody.

[0090] Besides the above method, in which a nonhuman animal is immunizedwith an antigen for preparing hybridoma, human lymphocytes such as thatinfected by EB virus may be immunized with a protein, protein expressingcells, or their lysates in vitro. Then, the immunized lymphocytes arefused with human-derived myeloma cells that is capable of indefinitelydividing, such as U266, to yield a hybridoma producing a desired humanantibody, able to bind to the protein can be obtained (UnexaminedPublished Japanese Patent Application (JP-A) No. Sho 63-17688).

[0091] Subsequently, the hybridomas thus obtained are transplanted intothe abdominal cavity of a mouse from which the ascites is collected. Themonoclonal antibodies thus obtained can be purified by, for example,ammonium sulfate precipitation or by column chromatography using aprotein A or protein G column, a DEAE ion exchange column, an affinitycolumn to which the protein of the invention is coupled, and such. Theantibody of the invention can be used not only for purifying anddetecting the protein of the invention, but also as a candidate for anagonist or antagonist to the protein of the present invention. It isalso expected to use the antibody for antibody therapy of diseasesassociated with the protein of this invention. When the antibodyobtained is administered to the human body (antibody therapy), humanantibodies or humanized antibodies are preferred to reduceimmunogenicity.

[0092] For example, transgenic animals having a repertory of humanantibody genes may be immunized with a protein, protein expressingcells, or their lysates as an antigen. Antibody producing cells arecollected from the animals, and fused with myeloma cells to obtainhybridoma, from which human antibodies against the protein can beprepared (see WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735,and WO96-34096).

[0093] Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

[0094] Monoclonal antibodies thus obtained can also be recombinantlyprepared using genetic engineering techniques (see, for example,Borrebaeck C. A. K. and Larrick J. W. Therapeutic Monoclonal Antibodies,published in the United Kingdom by MacMillan Publishers LTD (1990)). ADNA encoding an antibody may be cloned from an immune cell, such ashybridomas or immunized lymphocytes producing the antibody; insertedinto an appropriate vector; and introduced into host cells to prepare arecombinant antibody. The present invention also includes recombinantantibodies prepared as described above.

[0095] The antibody of the present invention may be a fragment of anantibody or modified antibody, so long as it binds to the protein of theinvention. For instance, the antibody fragment may be Fab, F(ab′)₂, Fv,or single chain Fv (scFv), in which Fv fragments from H and L chains areligated by an appropriate linker (Huston et al., Proc. Natl. Acad. Sci.U.S.A. 85:5879-5883, 1988). More specifically, an antibody fragment maybe generated by treating an antibody with an enzyme such as papain orpepsin. Alternatively, a gene encoding the antibody fragment may beconstructed; inserted into an expression vector; and expressed in anappropriate host cell (see, for example, Co et al., J. Immunol.152:2968-2976, 1994; Better et al., Methods Enzymol. 178:476-496, 1989;Pluckthun et al., Methods Enzymol. 178:497-515, 1989; Lamoyi, MethodsEnzymol. 121:652-663, 1986; Rousseaux et al., Methods Enzymol.121:663-669, 1986; Bird et al., Trends Biotechnol. 9:132-137, 1991).

[0096] An antibody may be modified by conjugation with a variety ofmolecules, such as polyethylene glycol (PEG). The antibody of thepresent invention includes such modified antibodies. A modified antibodycan be obtained by chemically modifying an antibody. These modificationmethods have been already established in the field.

[0097] Alternatively, the antibody of the present invention may beobtained as a chimeric antibody, between a variable region derived fromnonhuman antibody and the constant region derived from human antibody,or as a humanized antibody, comprising the complementarity determiningregion (CDR) derived from nonhuman antibody, the frame work region (FR)derived from human antibody, and the constant region. Such antibodiescan be prepared by using known technology.

[0098] Obtained antibodies may be purified to homogeneity. Theantibodies can be separated and purified by using standard methods forprotein separation and purification. For instance, column chromatographysuch as affinity chromatography, filter, ultrafiltration, saltprecipitation, dialysis, SDS-polyacrylamide gel electrophoresis,isoelectric point electrophoresis, and so on may be appropriatelyselected and combined to isolate and purify the antibody (Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988), but methods are not limited to them. Theconcentration of the antibody obtained as described above can bedetermined by the measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), or others.

[0099] Columns for affinity chromatography include protein A column andprotein G column. For example, protein A column includes Hyper D, POROS,Sepharose F.F. (Pharmacia) and the like.

[0100] In addition to affinity chromatography, chromatographic methodsinclude, for example, ion exchange chromatography, hydrophobicchromatography, gel filtration, reverse-phase chromatography, adsorptionchromatography and others (“Strategies for Protein Purification andCharacterization: A Laboratory Course Manual” Ed Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press, 1996). These chromatographicmethods can be conducted by using liquid chromatography such as HPLC andFPLC.

[0101] For example, absorbance measurement, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), orimmunofluorescence may be used to measure the antigen binding activityof the antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate; the protein of the invention isapplied to the plate; and then a sample containing a desired antibody,such as culture supernatant of antibody producing cells or purifiedantibodies, is applied. Then, a secondary antibody that recognizes theprimary antibody and which is labeled with an enzyme such as alkalinephosphatase is applied, and the plate is incubated. After washing, anenzyme substrate, such as p-nitrophenyl phosphate, is added to theplate, and the absorbance is measured to evaluate the antigen bindingactivity of the sample. A fragment of the protein, such as a C-terminalfragment, may be used as a protein. BIAcore (Pharmacia) may be used toevaluate the activity of the antibody according to the presentinvention.

[0102] The above methods allow for the detection or measurement of theprotein of the invention, by exposing the antibody of the invention to asample assumed to contain the protein of the invention, and detecting ormeasuring the immune complex formed by the antibody and the protein.Because the method of detection or measurement of the protein accordingto the invention can specifically detect or measure a protein, themethod may be useful in a variety of experiments in which the protein isused.

[0103] The present invention also provides a polynucleotide containingat least 15 nucleotides complementary to the DNA (SEQ ID NO:1) encodingthe human protein “C-NT2RP3001495” or the complementary strand thereof.

[0104] Herein, the term “complementary strand” is defined as one strandof a double strand DNA composed of A:T and G:C base pair to the otherstrand. Also, “complementary” is defined as not only those completelymatching within a continuous region of at least 15 nucleotides, but alsohaving a homology of at least 70%, favorably 80% or higher, morefavorably 90% or higher, and most favorably 95% or higher within thatregion. The homology may be determined using the algorithm describedherein.

[0105] Such a nucleic acid includes probes and primers used for thedetection and amplification of DNA encoding the inventive protein;probes and primers used for the detection of expression of the DNA; andnucleotide and nucleotide derivatives (e.g., antisense oligonucleotideand ribozyme, or DNAs encoding them, etc.) used for the regulation ofexpression of the inventive protein. In addition, such a nucleic acidcan also be used for the preparation of DNA chip.

[0106] When used as primers, such nucleic acids are complementary at the3′ end, and restriction enzyme recognition sequences or tags can beadded to the 5′ end.

[0107] The antisense oligonucleotides include, for example, antisenseoligonucleotides hybridizing to any region of the nucleotide sequence ofSEQ ID NO:1. The antisense oligonucleotide is preferably an antisense ofa continuous sequence of a length of 15 nucleotides or longer within thenucleotide sequence of SEQ ID NO:1. More preferably, the abovecontinuous sequence of a length of 15 nucleotides or longer contains thetranslation initiation codon.

[0108] A derivative or modified form of antisense oligonucleotide mayalso be used. The modified antisense oligonucleotides may be thosemodified with lower alkylphosphonate such as methylphosphonate andethylphosphonate; phosphorothioate; phosphoroamidate; and so on.

[0109] Herein, an antisense oligonucleotide is not restricted to thosein which all nucleotides are complementary to the correspondingnucleotides within a given region of a DNA or mRNA; so long as it canspecifically hybridize with the nucleotide sequences of SEQ ID NO:1, itmay have one or more nucleotide mismatches.

[0110] A derivative of the antisense oligonucleotide of the presentinvention may act on cells producing the protein of the invention andmay bind to a DNA or mRNA encoding the protein, whereby inhibiting theexpression of the protein of the invention by inhibiting itstranscription or translation, or by promoting the degradation of mRNA,and thereby inhibiting the function of the protein of the invention.

[0111] A derivative of the antisense oligonucleotide of the presentinvention may be mixed with appropriate carriers which are inactiveagainst the derivative, and may be used as a medicine for externallyapplication such as salve or poultice.

[0112] If necessary, it may be mixed with an excipient, isotonizingagent, solubilizing agent, stabilizer, preservative, pain-killer, or thelike, and prepared as a tablet, powder, granule, capsule, liposomecapsule, injectable solution, liquid formulation, nose drops,freeze-dried agent, etc. The above may be achieved according to standardmethods.

[0113] For treating patients, a derivative of an antisenseoligonucleotide of the present invention may be, for example, directlyapplied to the affected area of a patient, or administered into bloodvessels so as to finally reach the affected area. Moreover, thederivative may be encapsulated in antisense-encapsulating materials suchas liposome, poly-L-lysine, lipid, cholesterol, lipofectin, or theirderivative in order to increase durability and/or membrane permeability.

[0114] Dose of the derivative of the antisense oligonucleotide of thepresent invention may be appropriately adjusted depending on thepatient's conditions, and a favorable amount such as 0.1 to 100 mg/kg,or more preferably 0.1 to 50 mg/kg may be administered.

[0115] As the antisense oligonucleotides of the present inventioninhibit expression of the protein of the invention, they find utility asinhibitors of the biological activity of the protein of the invention.An inhibitor of expression comprising the antisense oligonucleotide ofthe present invention is useful because it can inhibit the biologicalactivity of the protein of the invention.

[0116] The protein of the invention may be used to screen for compoundsthat bind to the protein of the present invention. Specifically, theprotein may be used in methods of screening for compounds, which methodcomprises the steps of exposing the protein of the present invention toa test sample in which a compound binding to the protein is expected tobe contained; and selecting the compound having the activity of bindingto the protein.

[0117] The proteins of the invention used for screening may berecombinant or natural proteins, or partial peptides. Alternatively,they may be expressed on the surface of cells or in the form of amembrane fraction. There is no particular restriction on the test sampleas it includes, for example, cell extract, cell culture supernatant,product of fermentation microorganism, extract from marine organism,extract from plant, purified or crude protein, peptide, non-peptidecompound, synthetic low-molecular-weight compound, natural compound,etc. The inventive protein to be contacted with a test sample can becontacted with the test sample, for example, as a purified protein, as asoluble protein, in a form of protein immobilized on carriers, as afusion protein with other proteins, in a form of protein presented oncell membrane, as a membrane fraction.

[0118] Many methods known to those skilled in the art can be used toscreen proteins capable of binding to the inventive protein. Suchscreening can be carried out, for example, by the immunoprecipitationmethod. Specifically, the method can be carried out as follows. The geneencoding a protein of this invention is expressed by inserting the geneinto a vector for foreign gene expression in pSV2neo, pcDNA I, pCD8, andsuch, and expressing the gene in animal cells, etc. Any generally usedpromoters may be employed for the expression, including the SV40 earlypromoter (Rigby In Williamson (ed.), Genetic Engineering, Vol. 3.Academic Press, London, p.83-141 (1982)), EF-1 α promoter (Kim et al.,Gene 91:217-223, 1990), CAG promoter (Niwa et al., Gene 108:193-200,1991), RSV LTR promoter (Cullen, Methods in Enzymology 152:684-704,1987), SR a promoter (Takebe et al., Mol. Cell. Biol. 8:466, 1988), CMVimmediate early promoter (Seed et al., Proc. Natl. Acad. Sci. U.S.A.84:3365-3369, 1987), SV40 late promoter (Gheysen et al., J. Mol. Appl.Genet. 1:385-394, 1982), Adenovirus late promoter (Kaufman et al., Mol.Cell. Biol. 9:946, 1989), HSV TK promoter, etc.

[0119] Transfer of a foreign gene into animal cells for its expressioncan be performed by any of the following methods, including theelectroporation method (Chu et al., Nucl. Acid Res. 15:1311-1326, 1987),the calcium phosphate method (Chen et al., Mol. Cell. Biol. 7:2745-2752,1987), the DEAE dextran method (Lopata et al., Nucl. Acids Res.12:5707-5717, 1984; Sussman et al., Mol. Cell. Biol. 4:1642-1643, 1985),the lipofectin method (Derijard, Cell. 7:1025-1037, 1994; Lamb et al.,Nature Genetics 5:22-30, 1993; Rabindran et al., Science 259:230-234,1993), etc.

[0120] The protein of this invention can be expressed as a fusionprotein having a recognition site for a monoclonal antibody byintroducing the recognition site (epitope) for the monoclonal antibody,the specificity of which has been established, into the N- or C-terminusof the protein of this invention. For this purpose, commercialepitope-antibody systems can be utilized (Igaku, Experimental Medicine13:85-90, 1995). Vectors which can express fusion proteins with theβ-galactosidase, maltose-binding protein, glutathione S-transferase,green fluorescence protein (GFP), and such, via the multi-cloning siteare commercially available.

[0121] There is also a report that a fusion protein may be prepared byintroducing only small epitope portions consisting of several to a dozenamino acid residues so as not to change the property of the protein ofthe present invention by the fusion. For example, epitopes such aspolyhistidine (His-tag), influenza hemagglutinin (HA), human c-myc,FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10protein (T7-tag), human herpes simplex virus glycoprotein (HSV-tag),E-tag (epitope on the monoclonal phage), and such, and monoclonalantibodies to recognize them can be utilized as the epitope-antibodysystem for screening proteins binding to the protein of this invention(Igaku, Experimental Medicine 13:85-90, 1995).

[0122] In immunoprecipitation, immune complexes are formed by addingthese antibodies to the cell lysate prepared using suitable surfactants.The immune complex comprises a protein of this invention, a proteincomprising the binding ability with the protein, and an antibody.Immunoprecipitation can be also performed by using antibodies against aprotein of this invention, besides using antibodies against theabove-described epitopes. An antibody to a protein of this invention canbe prepared, for example, by inserting a gene encoding the protein ofthe invention into an appropriate expression vector of E. coli toexpress it in the bacterium, purifying the expressed protein, andimmunizing rabbits, mice, rats, goats, chicken, and such against thepurified protein. The antibody can be also prepared by immunizing theabove-described animals against synthetic partial peptides of theprotein of the present invention.

[0123] Immune complexes can be precipitated using, for example, ProteinA Sepharose and Protein G Sepharose when the antibody is a murine IgGantibody. In addition, if a protein of this invention is prepared as afusion protein with the epitope, such as GST, an immune complex can beformed by using a substance specifically binding to these epitopes, suchas glutathione-Sepharose 4B, in the same mannere as in the use of theantibody against the protein of the present invention.

[0124] Immune precipitation, in general, may be carried out accordingto, or following the method described in the literature (Harlow, E. andLane, D.: Antibodies, pp.511-552, Cold Spring Harbor Laboratorypublications, New York (1988)).

[0125] SDS-PAGE is generally used for the analysis of immunoprecipitatedproteins. Bound proteins can be analyzed based on the molecular weightsof proteins using a gel of an appropriate concentration. In this case,although proteins bound to a protein of this invention, in general, arehardly detectable by the usual protein staining method, such asCoomassie staining and silver staining, the detection sensitivity can beimproved by culturing cells in a medium containing radioisotopes, suchas ³⁵S-methionine and ³⁵S-cysteine, to label proteins inside the cells,and detecting the labeled proteins. Once the molecular weight of theprotein is determined, the desired protein can be purified directly fromthe SDS-polyacrylamide gel and can be sequenced.

[0126] In addition, proteins binding to a protein of this invention canbe isolated using the West-western blotting method (Skolnik et al., Cell65:83-90, 1991) with the protein of this invention. Namely, cDNA isisolated from cells, tissues, and organs, in which the protein bindingto a protein of this invention is expected to be expressed (e.g. liverand kidney), and transferred into a phage vector (for example, λgt11,ZAP, and such) to prepare a cDNA library, which is then expressed onLB-agarose plates. The protein thus expressed is fixed on a filter;reacted with the labeled, purified protein of this invention; andplaques expressing a protein bound to a protein of this invention can bedetected by the label. Methods for labeling the proteins of thisinvention include methods using the binding activity of biotin andavidin; methods using antibodies specifically binding to the proteins ofthis invention, or peptides or polypeptides fused with the protein ofthis invention (e.g., GST); methods using the radioisotopes; methodsusing fluorescence; etc.

[0127] Alternatively, in another embodiment of the method for screeningof the present invention, the two-hybrid system utilizing cells may beused (Fields et al., Trends Genet. 10:286-292, 1994; Dalton et al., Cell68:597-612, 1992; “MATCHMAKER Two-Hybrid System”, “Mammalian MATCHMAKERTwo-Hybrid Assay Kit”, “MATCHMAKER One-Hybrid System (all fromClontech), “HybriZAP Two-Hybrid Vector System” (Stratagene)). In thetwo-hybrid system, an inventive protein or a partial peptide thereof isfused with the SRF DNA-binding region or GAL4 DNA-binding region, andthen is expressed in yeast cells; a cDNA library, which express proteinsin the form of fusion protein with the VP16 or GAL4 transcriptionactivation region, is prepared from cells that are predicted to expressa protein binding to an inventive protein; the resulting cDNA library isintroduced into the above-mentioned yeast cells; and then a cDNA derivedfrom the library is isolated from a detected positive clone (when aprotein binding to the inventive protein is expressed in yeast cells,the reporter gene is activated by the binding of the two proteins, andthus positive clones are detectable). A protein encoded by the cDNA canbe prepared after the isolated cDNA is introduced and expressed in E.coli. Thus it is possible to prepare a protein binding to an inventiveprotein or the encoding gene. Reporter genes to be used in thetwo-hybrid system include, but are not limited to, for example, Ade2gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activatorinhibitor type 1) gene in addition to HIS3 gene. The screening by thetwo-hybrid method can be conduced by using mammalian cells or others inaddition to yeast.

[0128] Compounds binding to a protein of the present invention can bescreened by affinity chromatography. For example, a protein of theinvention is immobilized on a carrier of an affinity column, and a testsample, in which a protein binding to the protein of the invention issupposed to be expressed, is applied to the column. A test sample hereinmay be, for example, cell extracts, cell lysates, etc. After loading thetest sample, the column is washed, and proteins bound to a protein ofthe invention can be prepared.

[0129] The amino acid sequence of the resulting protein is thenanalyzed. Based on the result, an oligo-DNA is synthesized and used asthe probe to screen a cDNA library. This can provide a DNA encoding theprotein.

[0130] In the present invention, a biosensor on the basis of surfaceplasmon resonance phenomenon can be used as a means to detect or assaythe bound compounds. By utilizing the biosensor on the basis of surfaceplasmon resonance phenomenon, the interaction between the inventiveprotein and a test compound can be observed as a surface plasmonresonance signal in real time using a small amount of protein withoutlabeling (e.g., BIAcore, Pharmacia). Thus the binding between theinventive protein and the test compound can be assessed by usingbiosensor of BIAcore, or the like.

[0131] In addition, methods are known in the art for isolating compoundsbinding to a protein of the invention, which are not limited only toproteins (including agonists and antagonists). Such methods include, forexample, the method of screening for a molecule binding to a protein ofthe invention by contacting a synthetic compound or natural substancebank, or a random phage peptide display library with an immobilizedprotein of the invention, and the high-throughput screening method usinga combinatorial chemistry technique (Wrighton et al., Science273:458-64, 1996; Verdine G. L., Nature 384:11-13, 1996; Hogan J. C.Jr., Nature 384:17-9, 1996).

[0132] Compounds isolated by the screening of this invention arecandidates for agents to regulate the activity of a protein of thisinvention, and thought to be applied to treatments for disorders causedby expressional and functional abnormalities, and such of the protein,and diseases which can be treated by controlling the activity of theprotein. Compounds which can be obtained by the screening method of thisinvention, the partial structure of which is modified by addition,deletion and/or substitution, are also included in the compounds bindingto the protein of this invention.

[0133] When a protein of this invention or compounds isolated by thescreening of this invention are used as drugs for humans and otheranimals, for example, mice, rats, guinea pigs, rabbits, chickens, cats,dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, they canbe administered by directly administering the protein or isolatedcompound itself to a patient or by administering it after formulatedaccording to known pharmaceutical methods. They can be administered, asthe occasion demands, for example, orally, as sugar-coated tablets,capsules, elixirs and microcapsules, or parenterally, in the form ofsterile solutions in water or other pharmaceutically acceptable liquids,or suspensions for injections. For example, they may be formulated byappropriately mixing with pharmaceutically acceptable carriers or media,specifically sterile water, physiological saline, plant oil, emulsifyingagents, suspending agents, surfactants, stabilizers, seasonings,excipients, vehicles, anticeptics, binders, and such, in the unit dosageform required in a generally accepted pharmaceutical procedure. Amountsof effective ingredients in these pharmaceutical preparations areadjusted so as to obtain the appropriate dose in the specified range.

[0134] Additives which can be mixed in tablets and capsules include, forexample, binders such as gelatin, corn starch, tragacanth gum and arabicgum; excipients such as crystalline cellulose; bulking agents such ascorn starch, gelatin and alginic acid; lubricants such as magnesiumstearate; sweetening agents such as sucrose, lactose or saccharine; andflavors such as peppermint, Gaultheria adenothrix oil or cherry. Whenthe dispensing unit form is a capsule, liquid carriers, such as oil, canbe further added to the above-described materials. Sterile compositionsfor injection can be prescribed using vehicles such as distilled waterfor injection according to standard pharmaceutical procedure.

[0135] Aqueous solutions for injections include, for example,physiological saline, and isotonic solutions containing: glucose andother supplements such as D-sorbitol, D-mannose, D-mannitol, sodiumchloride, and such; and suitable solubilizers, for example, alcohols,more specifically, ethanol, polyalcohols such as propylene glycol,polyethylene glycol, non-ionic surfactants such as polysorbate 80 (TM)and HCO-50 may be used together.

[0136] Oily solutions, including sesame oil and soybean oil, and benzylbenzoate and benzyl alcohol may be used together as the solubilizer.Injections may be combined with buffers such as phosphate buffer andsodium acetate buffer; soothing agents such as procaine hydrochloride;stabilizers such as benzyl alcohol, phenols and antioxidants. Injectionsthus prepared are typically filled in suitable ampules.

[0137] The administration to patients is done by methods commonly knownto those skilled in the art, such as intraarterial, intravenous, orsubcutaneous injections, as well as intranasal, bronchial,intramuscular, percutaneous, or oral administrations. One skilled in theart can suitably select the dosage according to the body-weight or ageof a patient, or the method of administration. If the compound can beencoded by DNA, the DNA may be used for gene therapy by incorporatingthe DNA into a vector for gene therapy. Dosages and administrationmethods vary depending on the body-weight, age, symptoms, and such ofpatients, but those skilled in the art can appropriately select them.

[0138] Although the specific dosage of the protein of the inventionchanges according to the subject to be treated, the target organs,symptoms, and administration methods, it is generally considered to be,for example, about 100 μg to 20 mg one day for an adult (as body-weight60 kg) in the form of injections.

[0139] Though they vary depending on the symptoms, doses of compoundsbinding to a protein of this invention or compounds regulating theactivity of such a protein may be generally in the range of about 0.1 to100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to20 mg per day for adults (based on the body weight 60 kg) in the case oforal administration.

[0140] Though it varies depending on the subject to be administered,target organ, symptom and method of administration, a single dose of thecompounds for the parenteral administration is thought to be preferablyadministered, for example, when it is in the form of injection,intravenously to normal adults (based on the body weight 60 kg) in therange of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and morepreferably about 0.1 to 10 mg or thereabout per day. Doses converted onthe 60 kg body weight basis or the body surface area can be similarlyadministered to other animals.

[0141] All publications and patents cited herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

[0142]FIG. 1 is a schematic diagram showing the structural features ofthe amino acid sequences of human “C-NT2RP3001495” of the invention,chicken “12F08” and “HHCMA56”. The protein “C-NT2RP3001495” of thepresent invention contains WW domains and Adh short motif in theN-terminal region.

DETAILED DESCRIPTION

[0143] The invention is illustrated more specifically with reference tothe following examples, but is not to be construed as being limitedthereto.

EXAMPLE 1 Construction of a cDNA Library by the Oligo-Capping Method

[0144] The NT-2 neuron progenitor cells (Stratagene), teratocarcinomacells from human fetal testis, which can be differentiated into neuronsby the treatment with retinoic acid were cultured for two weeks afterinduction treatment by the addition of retinoic acid according to themanufacturer's instructions.

[0145] After the culture, the cells were collected, and mRNA wasextracted according to the method described in the literature (Sambrooket al., Molecular Cloning Second edition, Cold Spring harbor LaboratoryPress 1989). Then, poly(A)⁺ RNA was purified by using oligo dTcellulose.

[0146] This poly(A)⁺ RNA was used to construct a cDNA library by theoligo-capping method (Maruyama et al., Gene, 138:171-174, 1994). Usingthe Oligo-cap linker (agcaucgagu cggccuuguu ggccuacugg/SEQ ID NO:5) andthe Oligo-dT primer (gcggctgaag acggcctatg tggccttttt tttttttttt tt/SEQID NO:6), bacterial alkaline phosphatase (BAP) treatment, tobacco acidphosphatase (TAP) treatment, RNA ligation, the first strand cDNAsynthesis, and removal of RNA were performed according to the references(Suzuki et al., Protein, Nucleic acid and Enzyme 41:197-201, 1996;Suzuki et al., Gene 200:149-156, 1997). Then, 5′- and 3′-PCR primers(agcatcgagt cggccttgtt g/SEQ ID NO:7, and gcggctgaag acggcctatg t/SEQ IDNO:8, respectively) were used for performing PCR to convert the cDNAinto double stranded cDNA, which was then digested with SfiI. Then, theDraIII-cleaved pME18SFL3 (GenBank AB009864, expression vector) was usedfor cloning the cDNA in a unidirectional manner, and cDNA libraries wereobtained. The nucleotide sequence of the 5′- and 3′-ends of the cDNAclones was analyzed with a DNA sequencer (ABI PRISM 377, PE Biosystems)after sequencing reactions performed with the DNA sequencing reagents(Dye Terminator Cycle Sequencing FS Ready Reaction Kit, dRhodamineTerminator Cycle Sequencing FS Ready Reaction Kit, or BigDye TerminatorCycle Sequencing FS Ready Reaction Kit, PE Biosystems), according to theinstructions.

[0147] pME18SFL3 vector contains the SRα promoter and SV40 small tintron in the upstream, as well as the SV40 polyA addition signalsequence downstream of the cloning site, respectively. As the cloningsite of pME18SFL3 has asymmetrical DraIII sites, and the ends of cDNAfragments contain SfiI sites complementary to the DraIII sites, thecloned cDNA fragments can be unidirectionally inserted downstream of theSRα promoter. Therefore, clones containing full-length cDNA can beexpressed transiently by introducing the obtained plasmid directly intoCOS cells. Thus, the clones can be analyzed very easily in terms of theproteins that are the gene products of the clones, or in terms of thebiological activities of the proteins.

EXAMPLE 2 Estimation of the Completeness at the 5′-Ends of the ClonesContained in the cDNA Libraries Constructed by the Oligo-Capping Method

[0148] The full-length ratio at the 5′-end sequence of respective clonesin the human cDNA libraries constructed by the oligo-capping method wasdetermined as follows. The clones whose 5′-end sequences were consistentwith those of known human mRNA in the public database were judged to be“full-length” if they had a longer 5′-end sequence than that of theknown human mRNA; or even though the 5′-end sequence was shorter, if itcontained the translation initiation codon it was judged to have the“full-length” sequence. Clones which did not contain the translationinitiation codon were judged to be “not-full-length”. The full-lengthratio ((the number of full-length clones)/(the number of full-length andnot-full-length clones)) at the 5′-end of the cDNA clones from eachlibrary was determined by comparing with known human mRNA. As a result,the full-length ratio of the 5′-ends was 63.5%. The result indicatesthat the full-length ratio at the 5′-end sequence was extremely high inthe human cDNA clones obtained by the oligo-capping method.

EXAMPLE 3 Assessment of the Full-Length Ratio of the 5′-End of the cDNAby the ATGpr and the ESTiMateFL

[0149] The ATGpr, developed by Salamov A. A., Nishikawa T., andSwindells M. B. in the Helix Research Institute, is a program forprediction of the translation initiation codon based on thecharacteristics of the sequences in the vicinity of the ATG codon(Salamov et al., Bioinformatics 14:384-390, 1998;http://www.hri.cojp/atgpr/). The results are shown with expectations(also mentioned as ATGpr1 below) whether the ATG is a true initiationcodon (0.05-0.94). When the program was applied to the 5′-end sequencesof the clones from the cDNA library that was obtained by theoligo-capping method having 65% full-length ratio, the sensitivity andspecificity of the estimation of the full-length clone (clone containingthe N-terminus of the ORF) were improved to 82 to 83% by selecting onlyclones having an ATGpr1 score 0.6 or higher. The maximum ATGpr1 scorefor 5′-end sequence of NT2RP3001495 was 0.94.

[0150] Next, the ESTiMateFL was used for the assessment of the clones.The ESTiMateFL, developed by Nishikawa and Ota in the Helix ResearchInstitute, is a method for selecting clones expected to have afull-length cDNA by comparing with the 5′-end or 3′-end sequences ofESTs in the public database.

[0151] By this method, a cDNA clone is judged to be most likely not tobe full-length if there exist any ESTs which have longer 5′-end or3′-end sequences than the clone. The method is systematized for highthroughput analysis. A clone is judged to be full-length if the clonehas a longer 5′-end sequence than the ESTs in the public databasecorresponding thereto. Even if a clone has a shorter 5′-end, the cloneis judged to be full-length if the difference in length is within 50bases, and otherwise judged not to be full-length, for convenience.Those clones whose 5′-end sequence is matching with the known mRNA,about 80% of the clones judged to be full-length by the comparison withESTs were also judged to be full-length by the assessment of the 5′-endsequence by comparing with known mRNA. Also, about 80% of the clonesjudged to be not full-length in the 5′-end sequence by comparing withESTs were also judged to be not full-length in the 5′-end sequence bycomparison with known mRNA. The precision of the estimation by comparingwith ESTs is improved with increasing numbers of ESTs to be compared.However, in case with limited numbers of ESTs, the reliability becomeslow. Thus, the method is effective in excluding clones with highprobability of being not-full-length from the cDNA clones that issynthesized by the oligo-capping method having a 5′-end sequencefull-length ratio of about 60%. In particular, the ESTiMateFL isefficiently used in estimating the full-length ratio at the 3′-endsequence of cDNA of a human unknown mRNA, a significant number of whichare deposited in the public database as EST deposits.

[0152] Results of the above assessment for the full-length ratio showedthat the clone NT2RP3001495, of which maximal value of ATGpr1 is greaterthan 0.3, is a novel clone with a high probability of being full-lengthand also which shares no sequence identity with any of human ESTsequences at least either at the 5′-end sequence or 3′-end sequence, orboth ends.

EXAMPLE 4 Isolation of cDNA Associated with the Maintenance ofDifferentiation of Chicken Smooth Muscle Cells

[0153] New-laid eggs from chicken White Leghorn were incubated at 37° C.in an incubator. The fetuses were taken out after 15-day incubation. Thegizzard was resected and placed in a dish containing PBS (phosphatebuffer) with forceps. The resected gizzard was cut into small blockswith scissors, and then, was dispersed as individual cells bycollagenase treatment. Then, large cell aggregates were removed with afilter of 100 μm. The cells were washed with Dulbecco's modified Eagle'smedium (Nissui #05919) containing 20 mg/ml bovine serum albumin (BSA: 4SigmaA-7638), and the cell count was determined with a hemocytometer.5×10⁴ cells were plated on a 3.5-cm petri-dish, and were cultured at 37°C. overnight. These culture media were changed with 20 mg/ml BSA/DMEMcontaining 0.2 ng/ml insulin-like growth factor (IGF-I; BoehringerMannheim #1048066), and the media were replaced by fresh ones every twodays. The concentration of collagenase type-V (SigmaC-9263) was adjustedto 1 mg/ml by using Sol.3 (137 mM NaCl, 5 mM KCl, 4 mM NaHCO₃, 5.4 mMGlucose, 2 mM MgCl₂, 10 mM PIPES; adjusted to pH 6.5), and thecollagenase solution was used after sterilization by filtration. On theninth day of culture the cells were harvested, and then the total RNAwas extracted therefrom. The cells cultured with culture mediumcontaining IGF-I are called differentiated smooth muscle cells in theexperiments described below, while the cells that had been changed tohave a proliferative character by adding bovine serum or anti-IGF-Iantibody (Upstate Biotechnology #05-172) at a concentration of 5 μg/ml,are called dedifferentiated smooth muscle cells. The cells were notdedifferentiated by the addition of mouse IgG antibodies (hereinafterreferred to as control antibodies), which were not the anti-IGF-Iantibody but the subclass of which were the same as that of theanti-IGF-I antibody, and thus, the cells were maintained as thedifferentiated smooth muscle cells. Complementary DNAs were synthesizedfrom 1 μg of the total RNAs extracted from differentiated cells ordedifferentiated cells of chicken smooth muscle that had been preparedby adding the control antibodies or anti-IGF-I antibodies. The synthesisof the cDNAs was carried out by a method using CapFinede PCR SynthesisKit (CLONTECH #K1052-1) according to the manual thereof. Specifically,total RNAs used were prepared from three types of cells: chicken gizzardsmooth muscle cells dedifferentiated by adding neutralizing anti-IGF-Iantibodies (hereinafter abbreviated as CGSMC-B); differentiated smoothmuscle cells by the addition of 0.2 ng/ml IGF-I and the controlantibodies (hereinafter abbreviated as CGSMC-C); and dedifferentiatedsmooth muscle cells obtained one day after the addition of bovine serum(hereinafter abbreviated as CGSMC-D). A 3.5 μl solution containing 1 μgof total RNA, 1 μl of CDS primer (attached to the kit) and 0.5 μl ofCapSwitch II oligo (attached to the kit) were mixed, incubated at 70° C.for 2 minutes, and then was allowed to stand at room temperature. Then,according to the manual attached to the kit, 2 μl of 5× first strandbuffer, 1 μl of DTT, 1 μl of 10 mM dNTP and 1 μl of MMLV reversetranscriptase were further added to the mixture, and the resultingmixture was incubated at 42° C. for 1 hour. 40 μl of TE (pH 7.5) wasadded to the mixture, and then the resulting solution was incubated at72° C. for 7 minutes. A 1-μl aliquot of the resulting cDNA was diluted10-fold with distilled water, and was used for the PCR as describedbelow. The composition of the reaction mixture was as follows.

[0154] Composition of Reaction Mixture: 10 μl cDNA (10-fold dilution) 10μl 10× Advantage KlenTaq buffer  4 μl 2.5 mM dNTP  2 μl 10 μM PCR primer(attached to the kit)  2 μl Advantage KlenTaq mix (50×) 72 μl distilledwater

[0155] 0.2-ml PCR tubes containing the above-mentioned reactioncomponents were placed on a Thermal Cycler PE2400 (PE Biosystems)preheated to 95° C. After the denaturation at 95° C. for 1 minute, PCRwas conducted with 15 cycles of two steps: 95° C. for 15 seconds andthen 68° C. for five minutes. Then, PCR was further continued with thesame two-step profile, but a 15-μl aliquot was taken as a sample every 3cycles. The samples were used to identify the number of cycles where theproducts by PCR amplification had been increased logarithmically and, atthe same time, where the amplification had not been yet saturated. Theresults showed that the condition of 17 cycles may be reasonable, andthus PCR amplification was conducted under this condition by using 8tubes for each cDNA. The product of the PCR amplification in 8 tubes foreach cDNA were combined together, and then, were deproteinized by mixingwith an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1).Then, the solutions were concentrated by n-butanol extraction. Theconcentrated solutions were subjected to CLONTECH CHROMA SPIN-1000Column, and then, were eluted according to the manual by using 1×TNEbuffer (10 mM Tris-HCl (pH 8.0)/10 mM NaCl/0.1 mM EDTA). The subtractionwas carried out using the resulting cDNA according to the manual ofCLONTECH PCR-Select Subtraction Kit. 2 μg cDNA of CGSMC-B, CGSMC-C orCGSMC-D was digested with 15 units of restriction enzyme RsaI in a totalsolution volume of 50 μl by using a buffer attached to the kit at 37° C.for 3 hours. 2.5 μl of 20×EDTA/glycogen mix attached to the kit and 3volumes of SALT solution (attached to the kit) were added to themixture. The resulting mixture was vigorously mixed, and then 8 μl ofPCR-Pure BIND (attached to the kit) was added thereto. The mixedsolution was incubated at room temperature for five minutes, and thencentrifuged at 14,000 rpm for one minute. The supernatant was removed,and the precipitate was dissolved in 1 ml of WASH Solution (attached tothe kit) by pipetting. The solution was again centrifuged at 14,000 rpmfor one minute, and the supernatant was removed. Further, the residualWASH Solution was removed completely by repeating centrifugation. Afterair-drying for 10 minutes, the precipitate was suspended in 17 μl of TEbuffer by pipetting, and then the suspension was incubated at roomtemperature for five minutes while keeping the suspended state of thesolution. Then, the solution was centrifuged at 14,0000 rpm for fiveminutes. The supernatant containing eluted DNA was transferred to aseparate tube, and then 9 μl of 4 M ammonium acetate (NH₄OAc; attachedto the kit) and 75 μl of ethanol were added thereto for ethanolprecipitation. The tube was centrifuged for 20 minutes, and theresulting precipitate was washed with 80% ethanol. The precipitate wasair-dried for 10 minutes, and then was dissolved in 6.7 μl of 1×TNEbuffer. The sample of CGSMC-C, RsaI-digested cDNAs of whichconcentration was adjusted to 300 ng/μl, was used a tester, andtherefore, the sample was further subjected to adapter ligation. Thesample of CGSMC-C was incubated for ligation with adapter 1 (10 μM) oradapter 2 (10 μM) (both attached to the kit) by using 1 μl of T4 DNAligase in a total volume of 10 μl containing Ligation buffer attached tothe kit at 16° C. overnight. 1 μl of 20×EDTA/glycogen mix was added tothe mixture, and then the enzyme was inactivated by the treatment at 72°C. for five minutes. The resulting adapter-ligated tester cDNAs arecalled TC-1 and TC-2, respectively.

[0156] 1.5 μl of each dedifferentiated chicken gizzard smooth musclecells, CGSMC-B and CGSMC-D (300 ng/μl), which serve as driver cDNAdigested with restriction enzyme RsaI, were combined with 1.5 μl of TC-1and 1.5 μl of 4×Hybridization buffer attached to the kit to a totalvolume of 6 μl in a 0.2-ml tube. Then, one drop of mineral oil was addedthereto, and this is referred to as H1. Similarly, a tube was preparedwith TC-2 instead of TC-1, and was named H2. H1 and H2 were heated fordenaturation at 98° C. for 90 seconds in a Thermal cycler (PE BiosystemsPE2400), and then were incubated at 68° C. for 8 hours. A driver cDNAsolution with a total volume of 4 μl was freshly prepared by combining1.5 μl of dedifferentiated chicken gizzard smooth muscle cells, CGSMC-Band CGSMC-D (300 ng/μl), respectively, digested with restriction enzymeRsaI, and 1 μl of 4×Hybridization buffer attached to the kit. Thesolution was denatured by heat at 98° C. for 90 seconds. An air bubblewas sucked into the 200-μl pipette tip containing H2 to avoid directcontact of the solution with a solution of denatured driver cDNA thatwas sucked therein later. The solutions were transferred in a tubecontaining H1, and then were mixed by pipetting. The tube containing H1was allowed to stand on a Thermal cycler during the manipulation. Thetube was incubated at 68° C. overnight to hybridize the tester cDNA tothe cDNA in the driver cDNA. 200 μl of Dilution Buffer (attached to thekit) was added and mixed by pipetting, and the resulting mixture wasincubated at 75° C. for 7 minutes. This was stored as a dilutedsubtracted CGSMC IGF(+) cDNA at −20° C. The diluted subtracted CGSMCIGF(+) cDNA was subjected to primary PCR in a reaction solutioncontaining the following components.

[0157] Composition of Reaction Mixture: 16 μl distilled water 2.5 μl 10×Advantage KlenTaq PCR buffer 4 μl 2.5 mM dNTPs (TAKARA) 1 μl 10 μM PCRprimer 1 (attached to the kit) 0.5 μl 50× Advantage KlenTaq DNApolymerase 1 μl diluted subtracted CGSMC IGF (+) cDNA

[0158] These components were mixed in a 0.2-ml tube, and one drop ofmineral oil was added thereto. After incubation at 75° C. for fiveminutes, and then at 94° C. for 25 seconds, PCR was conducted with 27cycles of three steps: 94° C. for 10 seconds, 66° C. for 30 seconds, and72° C. for 90 seconds. 3 μl of the primary PCR product was diluted with27 μl of distilled water, and then the following PCR was conducted.

[0159] Composition of Reaction Mixture: 18.5 μl distilled water 2.5 μl10× Advantage KlenTaq PCR buffer 0.5 μl 10 mM dNTPs (attached to thekit) 1 μl 10 μM Nested primer 1 (attached to the kit) 1 μl 10 μM Nestedprimer 2 (attached to the kit) 0.5 μl 50× Advantage KlenTaq DNApolymerase 1 μl 10 times diluted primary PCR product

[0160] These components were mixed in a 0.2-ml tube, and one drop ofmineral oil was added thereto. After incubation at 94° C. for 25seconds, PCR was conducted with 19 cycles of three steps: 94° C. for 10seconds, 66° C. for 30 seconds, and 72° C. for 90 seconds. A sample wastaken from the tube at cycles 13, 15, 17, and 19, respectively, andthen, amplification of PCR products were tested by agarose gelelectrophoresis. According to the result, the products seemed to besaturated with more than 15 cycles. Thus, products obtained with 15cycles of PCR were used to carry out the following experiment. 8 μl outof a total reaction volume of 25 μl was used in the electrophoresis, andthe remaining 17 μl was combined with 5 volumes of buffer PB (QIAGEN;buffer attached to Qiaquick PCR purification kit). The resultingsolution was mixed well. This was loaded onto Qiaquick column (acomponent of the same kit as described above), and the column wascentrifuged at 13,000 rpm. 750 μl of Buffer PE (a component of the samekit as described above) was added to the column, and then the column wascentrifuged again. A fraction (5 μl) eluted with 30 μl of water attachedto the kit was used for TA cloning with a pGEM-T Vector system (A3600)from Promega according to the manual. After ligation at 4° C. overnight,2 μl of the reaction solution was used for the transformation of E. coliDH5α according to the procedure of the kit. The resulting colonies wereused for colony PCR using the above-mentioned Nested primer 1 and Nestedprimer 2. The resulting PCR products were purified by using MultiScreenfrom Millipore to remove the primers. Then DNA sequence analysis wasperformed by using respective primers and the Dye Terminator CycleSequencing FS Ready Reaction kit (Perkin Elmer; Catalog #402122). Thenucleotide sequence of the resulting cDNA fragment was determined, and asequence named “12F08” (SEQ ID NO:3) was obtained.

EXAMPLE 5 Isolation of Human Novel Gene “C-NT2RP3001495” Having Homologyto Chicken “12F08”

[0161] NCBI UniGene database was searched for homology to the cDNAfragment “12F08” (SEQ ID NO:3) obtained in Example 4 by NCBI BLASTN2.0.The result showed that the “12F08” sequence exhibited 82% homology to aclone Hs#S1388556 belonging to human Unigene cluster Hs.128045.Considering that the sequence comparison was made between chicken andhuman, the gene belonging to Unigene cluster Hs.128045 can be concludedto be the human orthologue to chicken 12F08. Then, the followingsequences belonging to Hs.128045 were assembled into a contig, and theobtained sequence was named Hs128045_(—)12F08con.

[0162] gnl|UG|Hs#S1388556 wb85d11.x1 Homo sapiens cDNA, 3′end/clone=IMAGE:2312469/clone_end=3′/gb=AI669330/gi=4834104

[0163] gnl|UG|Hs#S1579061 wr85e01.x1 Homo sapiens cDNA, 3′end/clone=IMAGE:2494488/clone_end=3′/gb=AI984802/gi=5812079

[0164] gnl|UG|Hs#S1008273 op61g03.1s1 Homo sapiens cDNA, 3′end/clone=IMAGE:1581364/clone_end=3′/gb=AA970231/gi=3145744

[0165] gnl|UG⊕Hs#S984806 ol41b06.s1 Homo sapiens cDNA, 3′end/clone=IMAGE:1526003/clone_end=3′/gb=AA912726/gi=3052118

[0166] The pfam motif database was searched for Hs128045_(—)12F08conusing estwisedb of the database search programs Wise2 designed by EwanBirney at Sanger Center. The result showed that both 12F08 andHs128045_(—)12F08con contained two WW domains. The WW domain is known tobe an important functional domain for protein-protein interaction, andmany proteins containing WW domain have been reported.

[0167] Then, cDNA sequences of the Helix Research Institute (helixclones; Japanese Patent Application No. Hei 11-248036; Japanese PatentApplication No. 2000-118776) were searched using the sequence obtainedfrom the above-mentioned Unigene Cluster as a query. The cDNA sequencesof the Helix Research Institute are clones obtained by the method inExamples 1 to 3 which probability of containing a full-length sequenceis high.

[0168] The homology search using the helix clones revealed that thesequence was identical to that of a helix clone “C-NT2RP3001495”. Inaddition, the gene has been identified to be identical to Hs.519 Humanoxidoreductase (HHCMA56) of Unigene as well. However, detailed analysisby using GAP of GCG Packaging software showed that “C-NT2RP3001495” waslonger than HHCMA56; the sequence of HHCMA56 started at nucleotide 578of “C-NT2RP3001495” sequence; the C-residue triplet at nucleotides 276to 278 in the sequence of HHCMA56 was altered to a C-residue doublet inthe sequence of “C-NT2RP3001495”; and further, the G-residue triplet atnucleotides 280 to 282 in the sequence of HHCMA56 was altered to aG-residue doublet at nucleotides 856 to 858 in the sequence of“C-NT2RP3001495”. Therefore, PCR amplification was performed using theoligonucleotide, 1495-588U24 (5′-GCA GGA ACA TGG CAA GGG CGA GTG-3′/SEQID NO:9), corresponding to the 24 nucleotides starting from nucleotide588 of “C-NT2RP3001495”, and the oligonucleotide, 1495-862L23 (5′-GGGCAG GAG CTG AGC GGC ACA AA-3′/SEQ ID NO:10), having a complementarystrand to the 23 nucleotides starting from nucleotide 839 of“C-NT2RP3001495”, as well as human genomic DNA (Clontech #6550-1) as thetemplate (pre-heating at 94° C. for 5 minutes; 45 cycles of denaturationat 94° C. for 15 seconds, annealing at 55° C. for 30 seconds, andextension at 72° C. for 30 seconds; final extension at 72° C. for 3minutes). The resulting DNA was cloned into pGEM-T (Promega; A3600), andthe sequence was determined.

[0169] As a result, it was revealed that the sequence of“C-NT2RP3001495” was correct, and the sequence of HHCMA56 containedincorrect nucleotides by misreading. Positional relation between theamino sequences encoded by respective genes is shown in FIG. 1. While anadh short motif, which is a motif found in oxidoreductase and dehydrase,is present in HHCMA56, the two WW domains, which are present in“C-NT2RP3001495”, are not found in HHCMA56. In addition, because of twonucleotide differences, HHCMA56 has been deposited as a gene encoding aprotein consisting of 371 amino acids, which is entirely different from“C-NT2RP3001495”. Thus, it can be stated that “C-NT2RP3001495” is anovel protein found for the first time by the present inventors. Theprotein “C-NT2RP3001495” is a protein consisting of 414 amino acidswhich contains two WW domain sequences, and is associated with themaintenance of differentiation of smooth muscle cells.

[0170] Furthermore, after the identification of the novel protein“C-NT2RP3001495” by the present inventors, a result of homology searchby BLASTN revealed that the sequence was identical to that depositedunder an accession number AF211943 (submitted on Dec. 7, 1999; publishedon May 5, 2000) in GenBank by Bednarek A K et al. (University of TexasMD Anderson Cancer Center). (WWOX, a novel WW domain-containing proteinmapping to human chromosome 16q23.3-24.1, a region frequently affectedin breast cancer, Cancer Res. 60(8): 2140-2145, 2000).

EXAMPLE 6 Expression Analysis of Chicken “12F08”

[0171] The expression level of the gene, chicken “12F08”, was analyzedby real-time PCR using ABI PRISM(R) 7700 Sequence Detection System fromPE Biosystems (PCR Meth. And Appl. 4:357-362, 1995). By usingoligonucleotides of sequence 8 (5′-GGT GGC TTT GCT GGA TTA TCT T-3′/SEQID NO:11) and sequence 9 (5′-GTT GCA GGA GGT CTG CCA TAT G-3′/SEQ IDNO:12), as well as G3PDH as an indicator, the expression levels of themRNA were compared with one another among chicken aorta (Ao), AoSMC cellderived from the aorta (AoDD), A (differentiated primary culture cell ofchicken gizzard smooth muscle), B (dedifferentiated primary culture cellof chicken gizzard smooth muscle by the addition of anti-IFG-Iantibody), C (differentiated primary culture cell of chicken gizzardsmooth muscle by the addition of control antibody), F1 (dedifferentiatedprimary culture cell of chicken gizzard smooth muscle one day after theaddition of FCS), Br (brain), Ca (cardiac muscle), Gz (gizzard), and Lv(liver). The total RNAs, which had been extracted from chicken tissuesof aorta (Ao), Br, Ca, Gz, and Lv directly obtained from a chicken, werekindly provided by Dr. Sobue at Osaka University. CAOMC (TC354-05) fromCell Applications, Inc. had been purchased from Toyobo. These cells werecultured according to the instructions in the protocol, and were used asAoSMC (AoDD) cells.

[0172] The results of real-time RT-PCR with ABI7700 are shown inTable 1. PCR was carried out with pre-heating at 95° C. for 10 minutes,and 50 cycles of denaturation at 94° C. for 20 seconds, annealing at 55°C. for 20 seconds, and extension at 72° C. for 30 seconds by using SYBRGreen PCR Core Reagent kit (PE Biosystems; 4304886). Each value isobtained by dividing the value of 12F08 expression level by the value ofexpression level of G3PDH as a control. The greater the value is, thehigher the expression level of 12F08 gene in the cells will be. Thechicken “12F08” was found to be expressed at a high level indifferentiated smooth muscle and gizzard. Accordingly, it was suggestedthat the chicken “12F08” gene encodes a protein participating in themaintenance of differentiation of smooth muscle cells. TABLE 1 TissueCells mRNA expression amount Ao 17.14 AoDD 0.88 A 3.43 B 2.37 C 8.4 F12.18 Br 4.45 Ca 2.36 Gz 6.41 Lv 1.35

EXAMPLE 7 Gene Expression Analysis by Hybridization Using High DensityDNA Filter

[0173] DNA for spotting onto the nylon membranes was prepared accordingto the following procedure. E. coli was cultured in each well of a96-well plate (in a LB medium at 37° C. for 16 hours). A part of eachculture was suspended in 10 μl of sterile water in the well of a 96-wellplate. The plate was heated at 100° C. for 10 minutes. Then the sampleswere analyzed by PCR. PCR was performed in a 20 μl solution per onereaction by using TaKaRa PCR Amplification Kit (Takara) according to thesupplier's protocol. A pair of sequencing primers, ME761FW (5′tacggaagtgttacttctgc 3′/SEQ ID NO:13) and ME1250RV (5′tgtgggaggttttttctcta 3′/SEQ ID NO:14), or a pair of primers, M13M4 (5′gttttcccagtcacgac 3′/SEQ ID NO:15) and M13RV (5′ caggaaacagctatgac 3′/SEQ ID NO:16) were used for the amplification of the insert cDNA inthe plasmid. PCR was performed in a thermal cycler, GeneAmp System 9600(PE Biosystems). The cycling profile consisted of pre-heating at 95° C.for 5 minutes; 10 cycles of denaturation at 95° C. for 10 seconds, andannealing/extension at 68° C. for 1 minute; 20 cycles of denaturation at98° C. for 20 seconds and annealing/extension at 60° C. for 3 minutes;and final extension at 72° C. for 10 minutes. After the PCR, 2 μl of thereaction solution was electrophoresed on a 1% agarose gel. DNA on thegel was stained with ethidium bromide to confirm the amplification ofcDNA. When cDNAs were not amplified by PCR, plasmids containing thecorresponding insert cDNAs were prepared by the alkali-extraction method(Sambrook et al., Molecular Cloning, A laboratory manual/2nd edition,Cold Spring Harbor Laboratory Press, 1989).

[0174] DNA array was prepared by the following procedure. An Aliquot ofthe DNA solution was added to each well of a 384-well plate. DNA wasspotted onto a nylon membrane (Boehringer) by using a 384-pin tool ofBiomek 2000 Laboratory Automation System (Beckman-Coulter). Morespecifically, the 384-well plate containing the DNA was placed under the384-pin tool. The independent 384 needles of the pin tool weresimultaneously dipped into the DNA solution to fix the DNA on theneedles. The needles were gently pressed onto a nylon membrane, and theDNA fixed on the needles was spotted onto the membrane. Denaturation ofthe spotted DNA and immobilization of the DNA on the nylon membrane werecarried out according to conventional methods (Sambrook et al.,Molecular Cloning, A laboratory manual/2nd edition, Cold Spring HarborLaboratory Press, 1989).

[0175] 1st strand cDNA labeled with radioisotope was used as thehybridization probe. The 1st strand cDNA was synthesized by usingThermoscript™ RT-PCR System (GIBCO). More specifically, the 1st strandcDNA was synthesized by using 1.5 μg mRNAs from various human tissues(Clontech), 1 μl 50 μM Oligo(dT)20, and 50 μCi [α³³P]dATP according tothe attached protocol. Purification of the probe was carried out byusing ProbeQuant™ G-50 micro column (Amersham-Pharmacia Biotech)according to the attached protocol. In the next step, 2 units of E. coliRNaseH were added to the reaction mixture. The mixture was incubated atroom temperature for 10 minutes, and then 100 μg of human COT-1 DNA(GIBCO) was added thereto. The mixture was incubated at 97° C. for 10minutes, and then was allowed to stand on ice to give the hybridizationprobe.

[0176] Hybridization of the radioisotope-labeled probe to the DNA arraywas performed in a usual manner (Sambrook et al., Molecular Cloning, Alaboratory manual/2nd edition, Cold Spring Harbor Laboratory Press,1989). The membrane was washed as follows: the nylon membrane was washedthree times by incubating in the Washing solution 1 (2×SSC, 1% SDS) atroom temperature (about 26° C.) for 20 minutes; then the membrane waswashed 3 times by incubating it in the Washing solution 2 (0.1×SSC, 1%SDS) at 65° C. for 20 minutes. Autoradiography was performed by using animage plate for BAS2000 (Fuji Photo Film Co., Ltd.). Specifically, thenylon membrane used for the hybridization was wrapped with a piece ofSaran Wrap, and was contacted with the light-sensitive surface of theimage plate. The membrane with the image plate was placed in an imagingcassette for radioisotope and was allowed to stand in dark for 4 hours.The radioactivity recorded on the image plate was analyzed by BAS2000(Fuji Photo Film Co., Ltd.) and was recorded as an image file of theautoradiogram by electronic conversion. The signal intensity of each DNAspot was analyzed by using Visage High Density Grid Analysis Systems(Genomic Solutions Inc.). The signal intensity was converted intonumerical data. The data were taken by duplicated measurements. Thereproducibility was assessed by comparing the signal intensities of thecorresponding spots on the duplicated DNA filters that were hybridizedto a single DNA probe. The ratio between the corresponding spots fallswithin a range of 2-folds or less in 95% of entire spots, and thecorrelation coefficient was r=0.97. Thus, the reproducibility wasassumed to be satisfactory.

[0177] The detection sensitivity in gene expression analysis wasestimated by examining increases in the signal intensity of the probeconcentration-dependent spot of the hybridization using a probecomplementary to the DNA spotted on the nylon membrane. PLACE1008092(the same DNA as that deposited in GenBank Accession No. AF107253) wasused as the DNA. The DNA array with the DNA of PLACE1008092 was preparedaccording to the above-mentioned method. The probe was prepared asfollows: mRNA was synthesized in vitro from the clone, PLACE1008092;using this mRNA as the template, radioisotope-labeled 1st strand cDNAwas synthesized in the same manner as the probe preparation methoddescribed above; and the cDNA was used as the probe. The cDNAPLACE1008092 was inserted into pBluescript SK(−), so that the 5′-end ofthe PLACE1008092 is ligated to the T7 promoter of the pBluescript SK(−)to give a recombinant plasmid for in vitro synthesis of the mRNA fromPLACE1008092. Specifically, the PLACE1008092 inserted at the DraIII siteof the pME18SFL3 was cut out by XhoI digestion. The resultingPLACE1008092 fragment was ligated to XhoI-predigested pBluescript SK(−)by using the DNA ligation kit ver.2 (Takara). The in-vitro mRNAsynthesis from PLACE1008092 inserted in pBluescript SK(−) was carriedout by using the Ampliscribe™ T7 high yield transcription kit (Epicentretechnologies). The hybridization and analysis of signal intensity ofeach DNA spot were conducted using the same methods described above.When the probe concentration was 1×10⁷ μg/ml or less, there was noincrease of signal intensity proportional to the probe concentration.Therefore it was assumed to be difficult to compare the signals with oneanother in this concentration range. Thus, spots with a intensity of 40or less were indiscriminately taken as low-level signals (FIG. 3).Within a concentration of the probe ranging from 1×10⁷ μg/ml to 0.1μg/ml, signals were found to increase in a probe concentration-dependentmanner. The detection sensitivity is 1:100,000 in a ratio of mRNAexpression level in a sample.

[0178] Table 2 shows the expression of each cDNA in human normal tissues(heart, lung, pituitary gland, thymus, brain, kidney, liver and spleen).The expression levels are indicated by numerical values of 0 to 10,000.The “C-NT2RP3001495” was expressed in at least one tissue. TABLE 2 ClonePituitary name Heart Lung gland Thymus Brain Kidney Liver Spleen GAPDH38.210 32.670 23.820 13.580 11.230 21.120 24.910 22.440 β-actin 279.280368.870 111.100 117.500 92.880 114.650 82.990 256.790 NT2RP3 42.34019.294 36.741 7.565 17.241 28.985 27.157 19.314 001495

EXAMPLE 8 Analysis of Genes Associated with Neural Cell Differentiation

[0179] Genes involved in neural cell differentiation are useful fortreating neurological diseases. It is possible that genes with varyingexpression levels in response to induction of cellular differentiationin neural cells are associated with neurological diseases. It wasexamined whether the expression of “C-NT2RP3001495” varies in responseto induction of differentiation (stimulation by retinoic acid (RA)) incultured cells of a neural strain, NT2.

[0180] The NT2 cells were treated basically according to the supplier'sinstruction manual. The term “undifferentiated NT2 cells” refers to NT2cells successively cultured in an OPTI-MEM I (GIBCO BRL; catalog No.31985) containing 10%(v/v) fetal bovine serum (GIBCO BRL) and 1%(v/v)penicillin-streptomycin (GIBCO BRL). The term “NT2 cells cultured in thepresence of retinoic acid” refers to cells passaged for 5 weeksfollowing transferring of the undifferentiated NT2 cells into a retinoicacid-containing medium, which consists of D-MEM (GIBCO BRL; catalog No.11965), 10%(v/v) fetal bovine serum, 1%(v/v) penicillin-streptomycin and10 μM retinoic acid (GIBCO BRL). The term “NT2 cells that were culturedin the presence of retinoic acid, and which were further cultured in amedia with the addition of cell-division inhibitor” refers to NT2 cellspassaged for 2 weeks following transferring of the NT2 cells cultured inthe presence of retinoic acid for 5 weeks into a cell-divisioninhibitor-containing medium, which consisted of D-MEM (GIBCO BRL;catalog No. 11965), 10%(v/v) fetal bovine serum, 1%(v/v)penicillin-streptomycin, 10 μM retinoic acid, 101M FudR(5-fluoro-2′-deoxyuridine: GIBCO BRL), 10 μM Urd (Uridine: GIBCO BRL)and 1 μM araC (Cytosine β-D-Arabinofuranoside: GIBCO BRL). Each of thecells were treated with trypsin and then were harvested. Total RNAs wereextracted from the cells by using S.N.A.P.™ Total RNA Isolation kit(Invitrogen). The probe used for hybridization was labeled by using 10μg of the total RNA according to the same methods as described above.

[0181] The data were obtained in triplicate (n=3). The data of signalvalue representing gene expression level in the cells in the presence ofstimulation for inducing differentiation were compared with thosewithout the stimulation. The comparison was performed by statisticaltreatment of two-sample t-test. Clones with significant difference inthe signal distribution were selected under the condition of p<0.05. Inthis analysis, clones with difference can be statistically detected evenwhen the signals are low. Accordingly, clones with signal value of 40 orless were also assessed.

[0182] Table 3 shows the expression level of “C-NT2RP3001495” cDNA inundifferentiated NT2 cells, NT2 cells cultured in the presence of RA,and NT2 cells cultured with the addition of cell-division inhibitorafter culturing in the presence of RA.

[0183] Averaged signal values (M₁, M₂) and sample variances (s₁ ², s₂ ²)were calculated for each gene in each of the cells, and then, the pooledsample variances s² were obtained from the sample variances of the twotypes of cells to be compared. The t values were determined according tothe following formula: t=(M₁−M₂)/s/(⅓+⅓)^(1/2). When the determinedt-value was greater than a t-value at P, the probability of significancelevel, of 0.05 or 0.01 in the t-distribution table with 4 degrees offreeedom, it was judged there exists a difference in the expressionlevel of the genes between the two types of cells at P<0.05 or P<0.01,respectively. The table also includes the information on an increase (+)or decrease (−) in the average expression level of a signal in theclones compared with that of undifferentiated cells.

[0184] As a result, the expression of the “C-NT2RP3001495” was shown toincrease by RA, suggesting that it is a clone involved in neurologicaldisorders. TABLE 3 NT2 NT2 RA NT2 RA INHIB ttest + ttest + Clone exp. 1exp. 2 exp. 3 exp. 1 exp. 2 exp. 3 exp. 1 exp. 2 exp. 3 N/R1 N/1 − GAPDH(Cr1) 3.53 1.08 0.98 2.92 2.49 2.8 1.76 2.59 1.52 βactin (Cr2) 155.381.18 99.68 148.45 110.68 101.34 114.68 105.79 151.13 NT2RP3001495 4.272.41 2.48 4.72 5.59 4.95 3.72 4.06 3.66 * +

EXAMPLE 9 Analysis of Rheumatoid Arthritis-Associated Genes

[0185] Proliferation of synovial cells covering inner surfaces of jointcavity and inflammatory reaction resulted from the action of cytokinesproduced by leukocytes infiltrating into the joint synovial tissues isthought to be involved in the onset of rheumatoid arthritis (JapanRheumatism Foundation Information Center, http://www.rheuma-net.or.jp/).Recent studies have also revealed that tissue necrosis factor (TNF)-αparticipates in the onset of rheumatoid arthritis (Current opinion inimmunology 1999, 11, 657-662). Those genes whose expression levelchanges in response to the action of TNF on synovial cells areconsidered to be involved in rheumatoid arthritis. It was examinedwhether the expression of “C-NT2RP3001495” varies in response to TNF-αin the primary cell culture of synovial tissue.

[0186] The primary cultured synovial cells (Cell Applications) weregrown to be confluent in a culture dish, and then, human TNF-α(Boehringer-Mannheim) was added at a final concentration of 10 ng/mlthereto. The culture was further continued for 24 hours. Total RNA wasextracted from the cells by using S.N.A.P.™ Total RNA Isolation kit(Invitrogen). The labeling of the probe used for hybridization wascarried out by using 10 μg of the total RNA according to the samemethods as described above. The data were obtained in triplicate (n=3).The data of signal value representing gene expression level in cellswith TNF stimulation were compared with those without the stimulation.The comparison was performed by statistical treatment of two-samplet-test. Clones with significant difference in the signal distributionwere selected under the condition of p<0.05. According to the analysis,clones with difference can be statistically detected even when thesignals were low. Accordingly, clones with signal value of 40 or lesswere also assessed for the selection.

[0187] Table 4 shows the expression level of each cDNA in synovial cellscultured under the absence or presence of TNF. Averaged signal values(M₁, M₂) and sample variances (s₁ ², s₂ ²) for each gene were calculatedin each of the cells, and then, the pooled sample variances s² wereobtained from the sample variances of the two types of cells to becompared. The t-values were determined according to the followingformula: t=(M₁−M₂)/s/(⅓+⅓)^(1/2). When the determined t-value wasgreater than a t-value at P, probability of significance level, of 0.05or 0.01 in the t-distribution table with 4 degrees of freedom, it wasjudged that a difference exists in the expression level of the genebetween the two types of cells at P<0.05 or P<0.01, respectively. Thetable also includes the information of an increase (+) or decrease (−)in the average expression level of a signal in the clones compared withthat of undifferentiated cells.

[0188] The results showed that the expression level of “C-NT2RP3001495”was reduced by TNF-α, suggesting that it is a clone associated withRheumatoid arthritis. TABLE 4 t test Synoviocyte Synoviocute_TNF vs +Clone exp. 1 exp. 2 exp. 3 exp. 1 exp. 2 exp. 3 TNF − GAPDH (Cr1) 0.40.8 0.89 0.9 1 1.15 βactin (Cr2) 385.94 262.23 582.98 443.28 422.61573.47 NT2RP3001495 4.14 4.14 3.85 2.75 2.92 1.76 * −

EXAMPLE 10 Analysis of Ultraviolet Radiation Damage-Associated Genes

[0189] It is known that ultraviolet rays give considerably adverseinfluence on health. In recent years, the risks of tissue damage byultraviolet rays has been increased due to the destruction of the ozonelayer, and ultraviolet radiation has been recognized as a risk factorfor diseases such as skin cancers (United States EnvironmentalProtection Agency: Ozone Depletion Home Page,http://www.epa.gov/ozone/). Genes whose expression levels change withexposure of the skin epidermal cells to ultraviolet rays are consideredto be associated with skin damage caused by ultraviolet radiation.Culturing primary cultured skin fibroblast cells irradiated withultraviolet ray, it was examined whether the expression of“C-NT2RP3001495” varies depending on the irradiation of ultraviolet ray.

[0190] First, after culturing to confluence in a culture dish, theprimary cultured skin fibroblast cells (Cell Applications) were exposedto 10,000 μJ/cm² of 254-nm ultraviolet light. Thereafter, messenger RNAswere extracted by using a FastTrack™ 2.0 mRNA Isolation kit (Invitrogen)from the unexposed cells and from the cells that were exposed to theultraviolet light and then cultured for 4 or 24 hours. The labeling ofthe hybridization probe was carried out by using 1.5 μg of each mRNA inthe same manner as described above. The data were obtained in triplicate(n=3). The hybridization signals were compared between the cells exposedto the ultraviolet light and the unexposed cells. The comparison waspreformed by statistical treatment with two-sample t-test. Clones withsignificant differences in the signal distribution were selected underthe condition of p<0.05. According to the analysis, the difference inthe signal values can be also detected statistically even when thesignal values are low. Accordingly, clones with signal value of 40 orlower were also assessed.

[0191] Table 5 shows the expression of each cDNA in skin-derivedfibroblast cells exposed and unexposed to ultraviolet light.

[0192] Averaged signal values (M₁, M₂) and sample variances (s₁ ², s₂ ²)were calculated for each gene in each of the cells, and then, pooledsample variances s² were obtained from the sample variances of the twotypes of cells to be compared. The t values were determined according tothe following formula: t=(M₁−M₂)/s/(⅓+⅓)^(1/2). When the determinedt-value was greater than a t-value at P, probability of significancelevel, of 0.05 or 0.01 in the t-distribution table with 4 degrees offreedom, it was judged that a difference exists in the expression levelof the gene between the two types of cells at P<0.05 or P<0.01,respectively. The table also includes the information of an increase (+)or decrease (−) in the average expression level of a signal in theclones compared with that of undifferentiated cells.

[0193] The results showed that the expression level of “C-NT2RP3001495”was reduced 4 hours or 24 hours after ultraviolet ray irradiation,suggesting that it is a clone associated with ultraviolet ray disorders.TABLE 5 UV_0 h UV_4 h UV_24 h t test 4 h 24 h Clone Exp. 1 Exp. 2 Exp. 3Exp. 1 Exp. 2 Exp. 3 Exp. 1 Exp. 2 Exp. 3 0/4 0/24 +/− +/− GAPDH (Cr1) 01.29 0.1 0.9 0.06 1.18 1.49 0.47 0 βactin (Cr2) 256.82 283.53 414.29388.38 117.29 329.8 189.18 190.26 157.87 * − NT2RP3001495 18.56 21.1119.03 12.08 9.76 9.93 15.89 18.33 21.35 ** −

INDUSTRIAL APPLICABILITY

[0194] The present invention provides a novel human protein“C-NT2RP3001495” associated with the maintenance of differentiation ofsmooth muscle cells and the gene encoding the protein. The protein hastwo WW domains that participate in protein-protein interaction. Thus, itis presumed that the protein regulates intracellular signaltransduction, gene expression and others through binding with otherproteins, and thereby participates in the maintenance of differentiationof smooth muscle cells. Abnormalities in the maintenance ofdifferentiation of smooth muscle cells have been known to cause avariety of diseases. For example, phenotypic modulation of vasculartunica media smooth muscle cell to a dedifferentiated type is recognizedin the early phases of the onset of arteriosclerosis and is known as themajor cause of thickening of vascular endothelium. Thus, the protein ofthe present invention participating in the maintenance ofdifferentiation of smooth muscle cells is considered to play importantroles in living body, and accordingly, it is useful as a target moleculein drug development. Further, compounds controlling functions of theinventive protein are expected to be pharmaceuticals for a variety ofdiseases caused by the abnormality in the maintenance of differentiationof smooth muscle cells, for example, ischemic heart diseases such asarteriosclerosis, myocardial infarction, aortic aneurysm, and cerebralapoplexy; cerebral vascular disorders; vascular dementia; as well asglomerulonephritis, pulmonary fibrosis, cerebral arteriosclerosis,hepatitis, and such, that are states of aberrant proliferation ofmesangial cells, alveolar epithelial cells, pericytes, and Ito cells,cells which have extremely similar characteristics to those of thesmooth muscle cell.

1 16 1 2256 DNA Homo sapiens CDS (125)...(1366) 1 cagtgcgcag gcgtgagcggtcgggccccg acgcgcgcgg gtctcgtttg gagcgggagt 60 gagttcctga gcgagtggacccggcagcgg gcgatagggg ggccaggtgc ctccacagtc 120 agcc atg gca gcg ctg cgctac gcg ggg ctg gac gac acg gac agt gag 169 Met Ala Ala Leu Arg Tyr AlaGly Leu Asp Asp Thr Asp Ser Glu 1 5 10 15 gac gag ctg cct ccg ggc tgggag gag aga acc acc aag gac ggc tgg 217 Asp Glu Leu Pro Pro Gly Trp GluGlu Arg Thr Thr Lys Asp Gly Trp 20 25 30 gtt tac tac gcc aat cac acc gaggag aag act cag tgg gaa cat cca 265 Val Tyr Tyr Ala Asn His Thr Glu GluLys Thr Gln Trp Glu His Pro 35 40 45 aaa act gga aaa aga aaa cga gtg gcagga gat ttg cca tac gga tgg 313 Lys Thr Gly Lys Arg Lys Arg Val Ala GlyAsp Leu Pro Tyr Gly Trp 50 55 60 gaa caa gaa act gat gag aac gga caa gtgttt ttt gtt gac cat ata 361 Glu Gln Glu Thr Asp Glu Asn Gly Gln Val PhePhe Val Asp His Ile 65 70 75 aat aaa aga acc acc tac ttg gac cca aga ctggcg ttt act gtg gat 409 Asn Lys Arg Thr Thr Tyr Leu Asp Pro Arg Leu AlaPhe Thr Val Asp 80 85 90 95 gat aat ccg acc aag cca acc acc cgg caa agatac gac ggc agc acc 457 Asp Asn Pro Thr Lys Pro Thr Thr Arg Gln Arg TyrAsp Gly Ser Thr 100 105 110 act gcc atg gaa att ctc cag ggc ccg gat ttcact ggc aaa gtg gtt 505 Thr Ala Met Glu Ile Leu Gln Gly Pro Asp Phe ThrGly Lys Val Val 115 120 125 gtg gtc act gga gct aat tca gga ata ggg ttcgaa acc gcc aag tct 553 Val Val Thr Gly Ala Asn Ser Gly Ile Gly Phe GluThr Ala Lys Ser 130 135 140 ttt gcc ctc cat ggt gca cat gtg atc ttg gcctgc agg aac atg gca 601 Phe Ala Leu His Gly Ala His Val Ile Leu Ala CysArg Asn Met Ala 145 150 155 agg gcg agt gaa gca gtg tca cgc att tta gaagaa tgg cat aaa gcc 649 Arg Ala Ser Glu Ala Val Ser Arg Ile Leu Glu GluTrp His Lys Ala 160 165 170 175 aag gta gaa gca atg acc ctg gac ctc gctctg ctc cgt agc gtg cag 697 Lys Val Glu Ala Met Thr Leu Asp Leu Ala LeuLeu Arg Ser Val Gln 180 185 190 cat ttt gct gaa gca ttc aag gcc aag aatgtg cct ctt cat gtg ctt 745 His Phe Ala Glu Ala Phe Lys Ala Lys Asn ValPro Leu His Val Leu 195 200 205 gtg tgc aac gca gca act ttt gct cta ccctgg agt ctc acc aaa gat 793 Val Cys Asn Ala Ala Thr Phe Ala Leu Pro TrpSer Leu Thr Lys Asp 210 215 220 ggc ctg gag acc acc ttt caa gtg aat catctg ggg cac ttc tac ctt 841 Gly Leu Glu Thr Thr Phe Gln Val Asn His LeuGly His Phe Tyr Leu 225 230 235 gtc cag ctc ctc cag gat gtt ttg tgc cgctca gct cct gcc cgt gtc 889 Val Gln Leu Leu Gln Asp Val Leu Cys Arg SerAla Pro Ala Arg Val 240 245 250 255 att gtg gtc tcc tca gag tcc cat cgattt aca gat att aac gac tcc 937 Ile Val Val Ser Ser Glu Ser His Arg PheThr Asp Ile Asn Asp Ser 260 265 270 ttg gga aaa ctg gac ttc agt cgc ctctct cca aca aaa aac gac tat 985 Leu Gly Lys Leu Asp Phe Ser Arg Leu SerPro Thr Lys Asn Asp Tyr 275 280 285 tgg gcg atg ctg gct tat aac agg tccaag ctc tgc aac atc ctc ttc 1033 Trp Ala Met Leu Ala Tyr Asn Arg Ser LysLeu Cys Asn Ile Leu Phe 290 295 300 tcc aac gag ctg cac cgt cgc ctc tcccca cgc ggg gtc acg tcg aac 1081 Ser Asn Glu Leu His Arg Arg Leu Ser ProArg Gly Val Thr Ser Asn 305 310 315 gca gtg cat cct gga aat atg atg tactcc aac att cat cgc agc tgg 1129 Ala Val His Pro Gly Asn Met Met Tyr SerAsn Ile His Arg Ser Trp 320 325 330 335 tgg gtg tac aca ctg ctg ttt accttg gcg agg cct ttc acc aag tcc 1177 Trp Val Tyr Thr Leu Leu Phe Thr LeuAla Arg Pro Phe Thr Lys Ser 340 345 350 atg caa cag gga gct gcc acc accgtg tac tgt gct gct gtc cca gaa 1225 Met Gln Gln Gly Ala Ala Thr Thr ValTyr Cys Ala Ala Val Pro Glu 355 360 365 ctg gag ggt ctg gga ggg atg tacttc aac aac tgc tgc cgc tgc atg 1273 Leu Glu Gly Leu Gly Gly Met Tyr PheAsn Asn Cys Cys Arg Cys Met 370 375 380 ccc tca cca gaa gct cag agc gaagag acg gcc cgg acc ctg tgg gcg 1321 Pro Ser Pro Glu Ala Gln Ser Glu GluThr Ala Arg Thr Leu Trp Ala 385 390 395 ctc agc gag agg ctg atc caa gaacgg ctt ggc agc cag tcc ggc 1366 Leu Ser Glu Arg Leu Ile Gln Glu Arg LeuGly Ser Gln Ser Gly 400 405 410 taagtggagc tcagagcgga tgggcacacacacccgccct gtgtgtgtcc cctcacgcaa 1426 gtgccagggc tgggcccctt ccaaatgtccctccaacaca gatccgcaag agtaaaggaa 1486 ataagagcag tcacaacaga gtgaaaaatcttaagtacca atgggaagca gggaattcct 1546 ggggtaaagt atcacttttc tggggctgggctaggcatag gtctctttgc tttctggtgg 1606 tggcctgttt gaaagtaaaa acctgcttggtgtgtaggtt ccgtatctcc ctggagaagc 1666 accagcaatt ctctttcttt tactgttatagaatagcctg aggtcccctc gtcccatcca 1726 gctaccacca cggccaccac tgcagccgggggctggcctt ctcctactta gggaagaaaa 1786 agcaagtgtt cactgctcct tgctgcattgatccaggaga taattgtttc attcatcctg 1846 accaagactg agccagctta gcaactgctggggagacaaa tctcagaacc ttgtcccagc 1906 cagtgaggat gacagtgaca cccagagggagtagaatacg cagaactacc aggtggcaaa 1966 gtacttgtca tagactcctt tgctaatgctatgcaaaaaa ttctttagag attataacaa 2026 atttttcaaa tcattcctta gataccttgaaaggcaggaa gggaagcgta tatacttaag 2086 aatacacagg atattttggg gggcagagaataaaacgtta gttaatccct ttgtctgtca 2146 atcacagtct cagttctctt gctttcacattgtacttaaa cctcctgctg tgcctcgcat 2206 cctatgctta ataaaagaac atgcttgaatatcaaaaaaa aaaaaaaaac 2256 2 414 PRT Homo sapiens 2 Met Ala Ala Leu ArgTyr Ala Gly Leu Asp Asp Thr Asp Ser Glu Asp 1 5 10 15 Glu Leu Pro ProGly Trp Glu Glu Arg Thr Thr Lys Asp Gly Trp Val 20 25 30 Tyr Tyr Ala AsnHis Thr Glu Glu Lys Thr Gln Trp Glu His Pro Lys 35 40 45 Thr Gly Lys ArgLys Arg Val Ala Gly Asp Leu Pro Tyr Gly Trp Glu 50 55 60 Gln Glu Thr AspGlu Asn Gly Gln Val Phe Phe Val Asp His Ile Asn 65 70 75 80 Lys Arg ThrThr Tyr Leu Asp Pro Arg Leu Ala Phe Thr Val Asp Asp 85 90 95 Asn Pro ThrLys Pro Thr Thr Arg Gln Arg Tyr Asp Gly Ser Thr Thr 100 105 110 Ala MetGlu Ile Leu Gln Gly Pro Asp Phe Thr Gly Lys Val Val Val 115 120 125 ValThr Gly Ala Asn Ser Gly Ile Gly Phe Glu Thr Ala Lys Ser Phe 130 135 140Ala Leu His Gly Ala His Val Ile Leu Ala Cys Arg Asn Met Ala Arg 145 150155 160 Ala Ser Glu Ala Val Ser Arg Ile Leu Glu Glu Trp His Lys Ala Lys165 170 175 Val Glu Ala Met Thr Leu Asp Leu Ala Leu Leu Arg Ser Val GlnHis 180 185 190 Phe Ala Glu Ala Phe Lys Ala Lys Asn Val Pro Leu His ValLeu Val 195 200 205 Cys Asn Ala Ala Thr Phe Ala Leu Pro Trp Ser Leu ThrLys Asp Gly 210 215 220 Leu Glu Thr Thr Phe Gln Val Asn His Leu Gly HisPhe Tyr Leu Val 225 230 235 240 Gln Leu Leu Gln Asp Val Leu Cys Arg SerAla Pro Ala Arg Val Ile 245 250 255 Val Val Ser Ser Glu Ser His Arg PheThr Asp Ile Asn Asp Ser Leu 260 265 270 Gly Lys Leu Asp Phe Ser Arg LeuSer Pro Thr Lys Asn Asp Tyr Trp 275 280 285 Ala Met Leu Ala Tyr Asn ArgSer Lys Leu Cys Asn Ile Leu Phe Ser 290 295 300 Asn Glu Leu His Arg ArgLeu Ser Pro Arg Gly Val Thr Ser Asn Ala 305 310 315 320 Val His Pro GlyAsn Met Met Tyr Ser Asn Ile His Arg Ser Trp Trp 325 330 335 Val Tyr ThrLeu Leu Phe Thr Leu Ala Arg Pro Phe Thr Lys Ser Met 340 345 350 Gln GlnGly Ala Ala Thr Thr Val Tyr Cys Ala Ala Val Pro Glu Leu 355 360 365 GluGly Leu Gly Gly Met Tyr Phe Asn Asn Cys Cys Arg Cys Met Pro 370 375 380Ser Pro Glu Ala Gln Ser Glu Glu Thr Ala Arg Thr Leu Trp Ala Leu 385 390395 400 Ser Glu Arg Leu Ile Gln Glu Arg Leu Gly Ser Gln Ser Gly 405 4103 251 DNA Gallus gallus CDS (3)...(251) 3 ag gag cgc acc acc aag gac ggctgg gtt tac tac gcc aat cac ttg 47 Glu Arg Thr Thr Lys Asp Gly Trp ValTyr Tyr Ala Asn His Leu 1 5 10 15 gaa gaa aaa aca cag tgg gaa cat ccaaaa tct ggg aag agg aaa cgt 95 Glu Glu Lys Thr Gln Trp Glu His Pro LysSer Gly Lys Arg Lys Arg 20 25 30 gtt gca gga ggt ctg cca tat gga tgg gagcag gag act gat gaa aat 143 Val Ala Gly Gly Leu Pro Tyr Gly Trp Glu GlnGlu Thr Asp Glu Asn 35 40 45 gga cag gtc tat ttt gta gac cac ata aac aaaaga act acc tat ctg 191 Gly Gln Val Tyr Phe Val Asp His Ile Asn Lys ArgThr Thr Tyr Leu 50 55 60 gat cca aga ttg gcc ttt aca gtt gaa gat aat ccagca aag cca cct 239 Asp Pro Arg Leu Ala Phe Thr Val Glu Asp Asn Pro AlaLys Pro Pro 65 70 75 act aga caa aaa 251 Thr Arg Gln Lys 80 4 83 PRTGallus gallus 4 Glu Arg Thr Thr Lys Asp Gly Trp Val Tyr Tyr Ala Asn HisLeu Glu 1 5 10 15 Glu Lys Thr Gln Trp Glu His Pro Lys Ser Gly Lys ArgLys Arg Val 20 25 30 Ala Gly Gly Leu Pro Tyr Gly Trp Glu Gln Glu Thr AspGlu Asn Gly 35 40 45 Gln Val Tyr Phe Val Asp His Ile Asn Lys Arg Thr ThrTyr Leu Asp 50 55 60 Pro Arg Leu Ala Phe Thr Val Glu Asp Asn Pro Ala LysPro Pro Thr 65 70 75 80 Arg Gln Lys 5 30 RNA Artificial SequenceSynthetically generated oligonucleotide 5 agcaucgagu cggccuuguuggccuacugg 30 6 42 DNA Artificial Sequence Synthetically generatedprimer 6 gcggctgaag acggcctatg tggccttttt tttttttttt tt 42 7 21 DNAArtificial Sequence Synthetically generated primer 7 agcatcgagtcggccttgtt g 21 8 21 DNA Artificial Sequence Synthetically generatedprimer 8 gcggctgaag acggcctatg t 21 9 24 DNA Artificial SequenceSynthetically generated primer 9 gcaggaacat ggcaagggcg agtg 24 10 23 DNAArtificial Sequence Synthetically generated primer 10 gggcaggagctgagcggcac aaa 23 11 22 DNA Artificial Sequence Synthetically generatedprimer 11 ggtggctttg ctggattatc tt 22 12 22 DNA Artificial SequenceSynthetically generated primer 12 gttgcaggag gtctgccata tg 22 13 20 DNAArtificial Sequence Synthetically generated primer 13 tacggaagtgttacttctgc 20 14 20 DNA Artificial Sequence Synthetically generatedprimer 14 tgtgggaggt tttttctcta 20 15 17 DNA Artificial SequenceSynthetically generated primer 15 gttttcccag tcacgac 17 16 17 DNAArtificial Sequence Synthetically generated primer 16 caggaaacag ctatgac17

What is claimed is:
 1. An isolated nucleic acid of any one of (a) to (d)below: (a) a nucleic acid encoding a protein comprising the amino acidsequence of SEQ ID NO:2, (b) a nucleic acid comprising a coding regionin the nucleotide sequence of SEQ ID NO:1, (c) a nucleic acid encoding aprotein that comprises the amino acid sequence of SEQ ID NO:2, in whichone or more amino acids are replaced, deleted, inserted and/or added andthat is functionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO:2, and (d) a nucleic acid that hybridizes understringent conditions with the nucleic acid comprising the nucleotidesequence of SEQ ID NO:1, and that encodes a protein functionallyequivalent to the protein comprising the amino acid sequence of SEQ IDNO:2.
 2. An isolated nucleic acid encoding the amino acid sequence ofSEQ ID NO:2 or a fragment thereof.
 3. A vector into which the nucleicacid of claim 1 is inserted.
 4. A vector into which the nucleic acid ofclaim 2 is inserted.
 5. A transformant harboring the nucleic acid ofclaim
 1. 6. A transformant harboring the nucleic acid of claim
 2. 7. Atransformant harboring the vector of claim
 3. 8. A transformantharboring the vector of claim
 4. 9. A substantially purified polypeptideencoded by the nucleic acid of claim
 1. 10. A substantially purifiedpolypeptide encoded by the nucleic acid of claim
 2. 11. A method forproducing a polypeptide, the method comprising the steps of culturingthe transformant of claim 7 and recovering a polypeptide expressed fromthe transformant or the culture supernatant thereof.
 12. A method forproducing a polypeptide, the method comprising the steps of culturingthe transformant of claim 8 and recovering a polypeptide expressed fromthe transformant or the culture supernatant thereof.
 13. An antibodyagainst the polypeptide of claim
 9. 14. An antibody against thepolypeptide of claim
 10. 15. A polynucleotide that hybridizes with thenucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or thecomplementary strand thereof and that comprises at least 15 nucleotides.16. A method for screening for a compound that binds to the polypeptideof claim 9, the method comprising the steps of: (a) contacting a testsample with the polypeptide or a partial peptide thereof, (b) detectinga binding activity of the test sample to the polypeptide or the partialpeptide thereof, and (c) selecting a compound comprising the bindingactivity to the polypeptide or the partial peptide thereof.
 17. A methodfor screening for a compound that binds to the polypeptide of claim 10,the method comprising the steps of: (a) contacting a test sample withthe polypeptide or a partial peptide thereof, (b) detecting a bindingactivity of the test sample to the polypeptide or the partial peptidethereof, and (c) selecting a compound comprising the binding activity tothe polypeptide or the partial peptide thereof.